Methods for ameliorating infusion reactions

ABSTRACT

Certain embodiments of the invention provide methods of ameliorating an infusion reaction in a mammal in need thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of priority of U.S.application Ser. No. 62/437,537, filed Dec. 21, 2016, which applicationis herein incorporated by reference.

BACKGROUND

Infusion reactions may occur during and after the intravenousadministration of proteins, liposomes, micelles and other natural orsynthetic macromolecules, aggregates or nanoparticles, such asnanoparticles in the submicron size range. Animal studies have indicatedthat many of these reactions are associated with complement activationand stimulation of the innate immune system. For example, administrationof lipid nanoparticles can be associated with adverse reactions, such asa temporary but substantial elevation or drop in blood pressure.

Accordingly, there is a continued unmet medical need for newcompositions and methods for preventing and treating infusion reactions.

SUMMARY OF THE INVENTION

Accordingly, certain embodiments of the invention provide a method ofameliorating an infusion reaction associated with intravenousadministration of at least one lipid formulated therapeutic agent in amammal in need thereof (e.g., a human), comprising administering to themammal via injection a therapeutically effective amount of anonsteroidal anti-inflammatory (NSAID) prior to the at least one lipidformulated therapeutic agent being intravenously administered.

Certain embodiments of the invention provide a method of ameliorating aninfusion reaction associated with intravenous administration of at leastone lipid formulated therapeutic agent in a mammal in need thereof(e.g., a human), comprising administering to the mammal, in order, 1) atherapeutically effective amount of a nonsteroidal anti-inflammatory(NSAID); and 2) the at least one lipid formulated therapeutic agent,wherein the NSAID is administered via injection and the at least onelipid formulated therapeutic agent is administered intravenously.

Certain embodiments of the invention provide a method of treating aninfusion reaction associated with intravenous administration of at leastone lipid formulated therapeutic agent in a mammal in need thereof(e.g., a human), comprising administering to the mammal via injection atherapeutically effective amount of a nonsteroidal anti-inflammatory(NSAID) prior to the at least one lipid formulated therapeutic agentbeing intravenously administered.

Certain embodiments of the invention provide a method of treating aninfusion reaction associated with intravenous administration of at leastone lipid formulated therapeutic agent in a mammal in need thereof(e.g., a human), comprising administering to the mammal, in order, 1) atherapeutically effective amount of a nonsteroidal anti-inflammatory(NSAID); and 2) the at least one lipid formulated therapeutic agent,wherein the NSAID is administered via injection and the at least onelipid formulated therapeutic agent is administered intravenously.

Certain embodiments of the invention provide a method of treating adisease or condition in a mammal in need thereof, comprisingadministering to the mammal via injection a therapeutically effectiveamount of a nonsteroidal anti-inflammatory (NSAID) prior to at least onelipid formulated therapeutic agent being intravenously administered.

Certain embodiments of the invention provide a method of treating adisease or condition in a mammal in need thereof, comprisingadministering to the mammal, in order, 1) a therapeutically effectiveamount of a nonsteroidal anti-inflammatory (NSAID); and 2) at least onelipid formulated therapeutic agent, wherein the NSAID is administeredvia injection and the at least one lipid formulated therapeutic agent isadministered intravenously.

Certain embodiments of the invention provide a kit comprising anonsteroidal anti-inflammatory (NSAID) and at least one lipid formulatedtherapeutic agent, a container, and a package insert or label indicatingthe administration of the NSAID via injection prior to the intravenousadministration of the at least one lipid formulated therapeutic agent,for ameliorating an infusion reaction associated with the intravenousadministration of the at least one lipid formulated therapeutic agent.

Certain embodiments of the invention provide a nonsteroidalanti-inflammatory (NSAID) for use in ameliorating an infusion reactionassociated with intravenous administration of at least one lipidformulated therapeutic agent in a mammal.

Certain embodiments of the invention provide the use of a nonsteroidalanti-inflammatory (NSAID) in the preparation of a medicament forameliorating an infusion reaction associated with intravenousadministration of at least one lipid formulated therapeutic agent in amammal.

Certain embodiments of the invention provide a combination of anonsteroidal anti-inflammatory (NSAID) and at least one lipid formulatedtherapeutic agent for the prophylactic or therapeutic treatment of adisease or condition.

Certain embodiments of the invention provide the use of a combination ofa nonsteroidal anti-inflammatory (NSAID) and at least one lipidformulated therapeutic agent in the preparation of a medicament for thetreatment of a disease or condition in a mammal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The effect of LNP2 infusion (with and without ketorolac ordexamethasone) on the concentration of thromboxane B2 in plasma frommixed venous blood. Results are expressed as the mean+/−sem (n=4) ateach time point.

FIG. 2. The effect of LNP2 infusion (with and without ketorolac ordexamethasone) on mean arterial blood pressure in the conscious minipigduring the infusion and for the first 30 minutes thereafter. Results arereported in 30 second intervals and are expressed as the mean response(n=4) at each time point.

FIG. 3. The effect of LNP2 infusion (with and without ketorolac ordexamethasone) on mean arterial blood pressure in the conscious minipig,recorded for a 24-hour period. Results are reported in 30 minuteintervals and are expressed as the mean+/−sem (n=4).

DETAILED DESCRIPTION

Certain methods of the invention may be used to promote improved safetyand tolerability of lipid formulated therapeutic agent(s). For example,the present invention provides methods for treating, preventing,reducing the risk or likelihood of developing (e.g., reducing thesusceptibility to), and/or ameliorating an infusion reaction associatedwith the intravenous administration of at least one lipid formulatedtherapeutic agent in a mammal in need thereof (e.g., human, such as ahuman in need thereof), the method comprising administering to themammal via injection a therapeutically effective amount of anonsteroidal anti-inflammatory (NSAID) prior to the at least one lipidformulated therapeutic agent being intravenously administered.

As used herein, the term “infusion reaction” refers to a variety ofsymptoms which may sometimes occur during and after the intravenousadministration of proteins, liposomes, micelles and other natural orsynthetic macromolecules, aggregates or nanoparticles, such as lipidnanoparticles in the submicron range. Manifestations of such reactionsmay include, but are not limited to, tachycardia, bradycardia, dyspnea,hypotension, hypertension, chest pain, dysrhythmias, flushing,urticaria, generalized pruritis, fever, rigors and bronchospasm.

Associated laboratory abnormalities may include leukocytosis, increasesin acute phase reactants and increased levels of cytokines andthromboxane B2. The reaction may be an acute reaction or may be adelayed reaction. For example, as described in the Example, acutehypertensive effects were observed within minutes of starting theinfusion, whereas delayed hypotension was observed hours later (e.g.,8-9 hours after the start of the infusion). A given subject's infusionreaction may comprise a single symptom or multiple symptoms.Additionally, a subject may experience multiple episodes of a givensymptom after the start of an infusion. Generally, symptoms typicallysubside within about 24 hours after the start of the infusion.

Accordingly, in certain embodiments of the invention the infusionreaction comprises one or more symptoms selected from tachycardia,bradycardia, dyspnea, hypotension, hypertension, chest pain and/orpressure, dysrhythmias, flushing, urticaria, generalized pruritis,fever, rigors, bronchospasm, leukocytosis, increased acute phasereactants, increased levels of cytokines and increased levels ofthromboxane B2. In certain embodiments, the infusion reaction compriseshypertension and/or hypotension. In certain embodiments, the infusionreaction comprises hypertension followed by hypotension. In certainembodiments, the infusion reaction comprises an increase in plasmathromboxane B2 levels.

As used herein, the term “ameliorate” or “ameliorating” refers topreventing occurrence or recurrence of one or more symptoms of aninfusion reaction, alleviation of symptoms of an infusion reaction,diminishment of any direct or indirect pathological consequences of aninfusion reaction, decreasing the rate of progression of an infusionreaction, and/or lessening the severity of one or more symptoms of aninfusion reaction. Methods for detecting/measuring infusion reactionsymptoms are known in the art, for example, using methods described inthe Example. In certain embodiments, symptoms are compared to a controlmammal, such as a mammal intravenously administered a lipid formulatedtherapeutic agent without prior injection of a NSAID.

Certain embodiments of the invention provide methods for treating aninfusion reaction associated with the intravenous administration of atleast one lipid formulated therapeutic agent in a mammal in need thereof(e.g., human, such as a human in need thereof), the method comprisingadministering to the mammal via injection a therapeutically effectiveamount of a nonsteroidal anti-inflammatory (NSAID) prior to the at leastone lipid formulated therapeutic agent being intravenously administered.

Certain embodiments of the invention also provide methods of treating adisease or condition in a mammal in need thereof, comprisingadministering to the mammal via injection a therapeutically effectiveamount of a nonsteroidal anti-inflammatory (NSAID) prior to at least onelipid formulated therapeutic agent being intravenously administered.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention to alter thenatural course of the individual being treated, and can be performedeither for prophylaxis or during the course of clinical pathology.Desirable effects of treatment include, but are not limited to,preventing occurrence or recurrence of a disease or condition,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease or condition, decreasing therate of progression of the disease or condition, amelioration orpalliation of the disease/condition state, and remission or improvedprognosis.

In certain embodiments, the disease or condition is a Hepatitis B viral(HBV) infection. In certain embodiments, the disease or condition is aHBV and a Hepatitis D viral (HDV) infection. The term “Hepatitis Bvirus” (abbreviated as HBV) refers to a virus species of the genusOrthohepadnavirus, which is a part of the Hepadnaviridae family ofviruses, and that is capable of causing liver inflammation in humans.The term “Hepatitis D virus” (abbreviated as HDV) refers to a virusspecies of the genus Deltaviridae, which is capable of causing liverinflammation in humans.

The term “nucleic acid” as used herein refers to a polymer containing atleast two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) ineither single- or double-stranded form and includes DNA and RNA.“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides. Nucleic acids include nucleicacids containing known nucleotide analogs or modified backbone residuesor linkages, which are synthetic, naturally occurring, and non-naturallyoccurring, and which have similar binding properties as the referencenucleic acid. Examples of such analogs and/or modified residues include,without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides,and peptide-nucleic acids (PNAs). Additionally, nucleic acids caninclude one or more UNA moieties.

The term “nucleic acid” includes any oligonucleotide or polynucleotide,with fragments containing up to 60 nucleotides generally termedoligonucleotides, and longer fragments termed polynucleotides. Adeoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribosejoined covalently to phosphate at the 5′ and 3′ carbons of this sugar toform an alternating, unbranched polymer. DNA may be in the form of,e.g., antisense molecules, plasmid DNA, precondensed DNA, a PCR product,vectors, expression cassettes, chimeric sequences, chromosomal DNA, orderivatives and combinations of these groups. A ribooligonucleotideconsists of a similar repeating structure where the 5-carbon sugar isribose. RNA may be in the form, for example, of small interfering RNA(siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetricalinterfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viralRNA (vRNA), and combinations thereof. Accordingly, the terms“polynucleotide” and “oligonucleotide” refer to a polymer or oligomer ofnucleotide or nucleoside monomers consisting of naturally-occurringbases, sugars and intersugar (backbone) linkages. The terms“polynucleotide” and “oligonucleotide” also include polymers oroligomers comprising non-naturally occurring monomers, or portionsthereof, which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofproperties such as, for example, enhanced cellular uptake, reducedimmunogenicity, and increased stability in the presence of nucleases.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions), alleles, orthologs, SNPs, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic AcidRes., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)).

An “isolated” or “purified” DNA molecule or RNA molecule is a DNAmolecule or RNA molecule that exists apart from its native environment.An isolated DNA molecule or RNA molecule may exist in a purified form ormay exist in a non-native environment such as, for example, a transgenichost cell. For example, an “isolated” or “purified” nucleic acidmolecule or biologically active portion thereof, is substantially freeof other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. In one embodiment, an“isolated” nucleic acid is free of sequences that naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “unlocked nucleobase analogue” (abbreviated as “UNA”) refers toan acyclic nucleobase in which the C2′ and C3′ atoms of the ribose ringare not covalently linked. The term “unlocked nucleobase analogue”includes nucleobase analogues having the following structure identifiedas Structure A:

wherein R is hydroxyl, and Base is any natural or unnatural base suchas, for example, adenine (A), cytosine (C), guanine (G) and thymine (T).UNA include the molecules identified as acyclic 2′-3′-seco-nucleotidemonomers in U.S. Pat. No. 8,314,227.

Oligonucleotides may specifically hybridize to or may be complementaryto a target polynucleotide sequence. The terms “specificallyhybridizable” and “complementary” as used herein indicate a sufficientdegree of complementarity such that stable and specific binding occursbetween the DNA or RNA target and the oligonucleotide. It is understoodthat an oligonucleotide need not be 100% complementary to its targetnucleic acid sequence to be specifically hybridizable. In preferredembodiments, an oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target sequence interferes withthe normal function of the target sequence to cause a loss of utility orexpression therefrom, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or, in the case of in vitro assays,under conditions in which the assays are conducted. Thus, theoligonucleotide may include 1, 2, 3, or more base substitutions ascompared to the region of a gene or mRNA sequence that it is targetingor to which it specifically hybridizes.

The term “small-interfering RNA” or “siRNA” as used herein refers todouble stranded RNA (i.e., duplex RNA) that is capable of reducing orinhibiting the expression of a target gene or sequence (e.g., bymediating the degradation or inhibiting the translation of mRNAs whichare complementary to the siRNA sequence) when the siRNA is in the samecell as the target gene or sequence. The siRNA may have substantial orcomplete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). In certain embodiments, thesiRNAs may be about 19-25 (duplex) nucleotides in length, and ispreferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length.siRNA duplexes may comprise 3′ overhangs of about 1 to about 4nucleotides or about 2 to about 3 nucleotides and 5′ phosphate termini.Examples of siRNA include, without limitation, a double-strandedpolynucleotide molecule assembled from two separate stranded molecules,wherein one strand is the sense strand and the other is thecomplementary antisense strand.

Preferably, siRNA are chemically synthesized. siRNA can also begenerated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

The phrase “inhibiting expression of a target gene” refers to theability of a siRNA to silence, reduce, or inhibit expression of a targetgene (e.g., a gene within the HBV genome). To examine the extent of genesilencing, a test sample (e.g., a biological sample from an organism ofinterest expressing the target gene or a sample of cells in cultureexpressing the target gene) is contacted with a siRNA that silences,reduces, or inhibits expression of the target gene. Expression of thetarget gene in the test sample is compared to expression of the targetgene in a control sample (e.g., a biological sample from an organism ofinterest expressing the target gene or a sample of cells in cultureexpressing the target gene) that is not contacted with the siRNA.Control samples (e.g., samples expressing the target gene) may beassigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe value of the test sample relative to the control sample (e.g.,buffer only, an siRNA sequence that targets a different gene, ascrambled siRNA sequence, etc.) is about 100%, 99%, 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%,80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, Northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art. An“effective amount” or “therapeutically effective amount” of atherapeutic nucleic acid such as a siRNA is an amount sufficient toproduce the desired effect, e.g., an inhibition of expression of atarget sequence in comparison to the normal expression level detected inthe absence of a siRNA. In particular embodiments, inhibition ofexpression of a target gene or target sequence is achieved when thevalue obtained with a siRNA relative to the control (e.g., buffer only,an siRNA sequence that targets a different gene, a scrambled siRNAsequence, etc.) is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%,77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, 5%, or 0%. Suitable assays for measuring the expression of atarget gene or target sequence include, but are not limited to,examination of protein or mRNA levels using techniques known to those ofskill in the art, such as, e.g., dot blots, Northern blots, in situhybridization, ELISA, immunoprecipitation, enzyme function, as well asphenotypic assays known to those of skill in the art.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

The term “lipid particle” includes a lipid formulation that can be usedto deliver a therapeutic agent (e.g., a nucleic acid, such as a siRNA)to a target site of interest (e.g., cell, tissue, organ, and the like).In preferred embodiments, the lipid particle is typically formed from acationic lipid, a non-cationic lipid, and optionally a conjugated lipidthat prevents aggregation of the particle. A lipid particle thatincludes a therapeutic agent is referred to as a therapeutic agent-lipidparticle. A lipid particle that includes a nucleic acid molecule (e.g.,siRNA molecule) is referred to as a nucleic acid-lipid particle.Typically, the nucleic acid is fully encapsulated within the lipidparticle, thereby protecting the nucleic acid from enzymaticdegradation.

In certain instances, nucleic acid-lipid particles are extremely usefulfor systemic applications, as they can exhibit extended circulationlifetimes following intravenous (i.v.) injection, they can accumulate atdistal sites (e.g., sites physically separated from the administrationsite), and they can mediate silencing of target gene expression at thesedistal sites. The nucleic acid may be complexed with a condensing agentand encapsulated within a lipid particle as set forth in PCT PublicationNo. WO 00/03683, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The lipid particles typically have a mean diameter of from about 30 nmto about 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 nm to about 100 nm, from about 80 nm toabout 100 nm, from about 90 nm to about 100 nm, from about 70 to about90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm,75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and aresubstantially non-toxic. In addition, nucleic acids, when present in thelipid particles, are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and20070042031, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides a therapeutic nucleic acid such as a siRNA, with fullencapsulation, partial encapsulation, or both. In a preferredembodiment, the nucleic acid (e.g., siRNA) is fully encapsulated in thelipid particle (e.g., to form a nucleic acid-lipid particle).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, PEG-lipid conjugates such as, e.g., PEG coupled todialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled todiacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol,PEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids,polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates),polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.Additional examples of POZ-lipid conjugates are described in PCTPublication No. WO 2010/006282. PEG or POZ can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG or the POZ to a lipid can be usedincluding, e.g., non-ester containing linker moieties andester-containing linker moieties. In certain preferred embodiments,non-ester containing linker moieties, such as amides or carbamates, areused.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The terms “cationic lipid” and “amino lipid” are used interchangeablyherein to include those lipids and salts thereof having one, two, three,or more fatty acid or fatty alkyl chains and a pH-titratable amino headgroup (e.g., an alkylamino or dialkylamino head group). The cationiclipid is typically protonated (i.e., positively charged) at a pH belowthe pK_(a) of the cationic lipid and is substantially neutral at a pHabove the pK_(a). The cationic lipids may also be termed titratablecationic lipids. In some embodiments, the cationic lipids comprise: aprotonatable tertiary amine (e.g., pH-titratable) head group; C₁₈ alkylchains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1,2, or 3) double bonds; and ether, ester, or ketal linkages between thehead group and alkyl chains. Such cationic lipids include, but are notlimited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA,DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K),DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, γ-DLen-C2K-DMA,DLin-M-C2-DMA (also known as MC2), and DLin-M-C3-DMA (also known asMC3).

The term “salts” includes any anionic and cationic complex, such as thecomplex formed between a cationic lipid and one or more anions.Non-limiting examples of anions include inorganic and organic anions,e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g.,hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogenphosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride,bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogensulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate,acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate,gluconate, malate, mandelate, tiglate, ascorbate, salicylate,polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite,bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate,arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,hydroxide, peroxide, permanganate, and mixtures thereof. In particularembodiments, the salts of the cationic lipids disclosed herein arecrystalline salts.

Administration of a compound as a pharmaceutically acceptable acid orbase salt may be appropriate. Examples of pharmaceutically acceptablesalts are organic acid addition salts formed with acids which form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The term “alkyl” includes a straight chain or branched, noncyclic orcyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbonatoms. Representative saturated straight chain alkyls include, but arenot limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, andthe like, while saturated branched alkyls include, without limitation,isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, whileunsaturated cyclic alkyls include, without limitation, cyclopentenyl,cyclohexenyl, and the like.

The term “alkenyl” includes an alkyl, as defined above, containing atleast one double bond between adjacent carbon atoms. Alkenyls includeboth cis and trans isomers. Representative straight chain and branchedalkenyls include, but are not limited to, ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike.

The term “alkynyl” includes any alkyl or alkenyl, as defined above,which additionally contains at least one triple bond between adjacentcarbons. Representative straight chain and branched alkynyls include,without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

The term “acyl” includes any alkyl, alkenyl, or alkynyl wherein thecarbon at the point of attachment is substituted with an oxo group, asdefined below. The following are non-limiting examples of acyl groups:—C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl.

The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7- to10-membered bicyclic, heterocyclic ring which is either saturated,unsaturated, or aromatic, and which contains from 1 or 2 heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzenering. The heterocycle may be attached via any heteroatom or carbon atom.Heterocycles include, but are not limited to, heteroaryls as definedbelow, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” mean that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O), two hydrogen atoms are replaced.In this regard, substituents include, but are not limited to, oxo,halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y),—NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(Y),—SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0, 1, or 2, R^(x) andR^(Y) are the same or different and are independently hydrogen, alkyl,or heterocycle, and each of the alkyl and heterocycle substituents maybe further substituted with one or more of oxo, halogen, —OH, —CN,alkyl, —OR^(x), heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y),—NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(Y),—SO_(n)R^(x), and —SO_(n)NR^(x)R^(y). The term “optionally substituted,”when used before a list of substituents, means that each of thesubstituents in the list may be optionally substituted as describedherein.

The term “halogen” includes fluoro, chloro, bromo, and iodo.

The term “fusogenic” refers to the ability of a lipid particle to fusewith the membranes of a cell. The membranes can be either the plasmamembrane or membranes surrounding organelles, e.g., endosome, nucleus,etc.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

The term “electron dense core”, when used to describe a lipid particle,refers to the dark appearance of the interior portion of a lipidparticle when visualized using cryo transmission electron microscopy(“cyroTEM”). Some lipid particles have an electron dense core and lack alipid bilayer structure. Some lipid particles have an electron densecore, lack a lipid bilayer structure, and have an inverse Hexagonal orCubic phase structure. While not wishing to be bound by theory, it isthought that the non-bilayer lipid packing provides a 3-dimensionalnetwork of lipid cylinders with water and nucleic acid on the inside,i.e., essentially a lipid droplet interpenetrated with aqueous channelscontaining the nucleic acid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles means thatthe particle is not significantly degraded after exposure to a serum ornuclease assay that would significantly degrade free DNA or RNA.Suitable assays include, for example, a standard serum assay, a DNAseassay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent suchas a siRNA within an organism. Some techniques of administration canlead to the systemic delivery of certain agents, but not others.Systemic delivery means that a useful, preferably therapeutic, amount ofan agent is exposed to most parts of the body. To obtain broadbiodistribution generally requires a blood lifetime such that the agentis not rapidly degraded or cleared (such as by first pass organs (liver,lung, etc.) or by rapid, nonspecific cell binding) before reaching adisease site distal to the site of administration. Systemic delivery oflipid particles can be by any means known in the art including, forexample, intravenous, subcutaneous, and intraperitoneal. In a preferredembodiment, systemic delivery of lipid particles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentsuch as a siRNA directly to a target site within an organism. Forexample, an agent can be locally delivered by direct injection into adisease site, other target site, or a target organ such as the liver,heart, pancreas, kidney, and the like.

The term “virus particle load”, as used herein, refers to a measure ofthe number of virus particles (e.g., HBV and/or HDV) present in a bodilyfluid, such as blood. For example, particle load may be expressed as thenumber of virus particles per milliliter of, e.g., blood. Particle loadtesting may be performed using nucleic acid amplification based tests,as well as non-nucleic acid-based tests (see, e.g., Puren et al., TheJournal of Infectious Diseases, 201:S27-36 (2010)).

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

Nonsteroidal Anti-Inflammatory Drugs (NSAIDs)

As discussed herein, certain embodiments of the invention providemethods for ameliorating an infusion reaction associated withintravenous administration of at least one lipid formulated therapeuticagent in a mammal in need thereof (e.g., a human subject), comprisingadministering to the mammal via injection a therapeutically effectiveamount of a nonsteroidal anti-inflammatory (NSAID) prior to the at leastone lipid formulated therapeutic agent being intravenously administered.

NSAIDs are a drug class that provide analgesic (pain-killing) andantipyretic (fever-reducing) effects, and, in higher doses,anti-inflammatory effects. The term nonsteroidal distinguishes thesedrugs from steroids, which, among a broad range of other effects, have asimilar eicosanoid-depressing, anti-inflammatory action. Typically,NSAIDs inhibit the activity of cyclooxygenase-1 (COX-1) andcyclooxygenase-2 (COX-2), and thereby the synthesis of prostaglandinsand thromboxanes.

NSAIDs include for example, but are not limited to, aspirin(acetylsalicylic acid), diflunisal, salicylic acid, salicylates,salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen,dexketoprofen, fluibiprofen, oxaprozin, loxoprofen, indomethacin,tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac,nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam,isoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamicacid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib,lumiracoxib, etoricoxib, firocoxib, nimesulide, clonixin, licofelone,flunixin meglumin and H-harpagide.

In certain embodiments, the NSAID is a NSAID that is capable of beingadministered via injection (e.g., parenterally, such as intravenously,intramuscularly or subcutaneously). In certain embodiments, the NSAID isa NSAID that is capable of being administered parenterally. In certainembodiments, the NSAID is a NSAID that is capable of being administeredintravenously. In certain embodiments, the NSAID is a NSAID discussed inPain Medicine, 2013; 14:S11-S17, which is incorporated by reference inits entirety herein.

In certain embodiments, the NSAID is selected from the group consistingof indomethacin, ketorolac, ibuprofen, diclofenac, lornoxicam,parecoxib, tenoxicam, phenylbutazone and flunixin meglumin. In certainembodiments, the NSAID is selected from the group consisting ofindomethacin, ketorolac, ibuprofen, diclofenac, lornoxicam, parecoxib,tenoxicam. In certain embodiments, the NSAID is selected from the groupconsisting of indomethacin, ketorolac and ibuprofen. In certainembodiments, the NSAID is ketorolac. In certain embodiments, the NSAIDis indomethacin. In certain embodiments, the NSAID is ibuprofen.

As discussed herein, the NSAID is generally administered via injection.Accordingly, in certain embodiments, the NSAID is administeredparenterally. In certain embodiments, the NSAID is administeredintravenously. In certain embodiments, the NSAID is administeredintramuscularly. In certain embodiments, the NSAID is administeredsubcutaneously.

Therapeutic Agents

As discussed herein, injection of a NSAID prior to intravenousadministration of at least one lipid formulated therapeutic agent mayameliorate an infusion reaction. Accordingly, in certain embodiments,the therapeutic agent is an agent that is capable of being formulated ina lipid (e.g., in a lipid nanoparticle formulation (LNP)).

Additionally, certain embodiments of the invention also provide for theadministration of one or more additional therapeutic agents (i.e., asecond, third, fourth, etc. therapeutic agent), which may or may not belipid formulated. In certain embodiments, the one or more additionaltherapeutic agents are administered sequentially or simultaneously withthe NSAID or the lipid formulated therapeutic agent. For example, incertain embodiments, dexamethasone may be administered sequentially orsimultaneously with the NSAID.

Thus, the therapeutic agent (i.e., the lipid formulated therapeuticagent or the additional therapeutic agent) may be of natural orsynthetic origin. For example, it may be a nucleic acid, a polypeptide,a protein, a peptide, or an organic compound. In certain embodiments,the therapeutic agent is a nucleic acid, a polypeptide or an organiccompound. In one embodiment, the therapeutic agent is a siRNA, mRNA, asmall molecule or an antibody.

In certain embodiments, the therapeutic agent is a polypeptide, forexample, an antibody, or a fragment or derivative thereof, such as a Fabfragment, a CDR region, or a single chain antibody. In certainembodiments, the therapeutic agent is an anti-PD-1 antibody, or fragmentthereof.

In certain embodiments, the therapeutic agent is a small molecule. Theterm “small molecule” includes organic molecules having a molecularweight of less than about 1000 amu. In one embodiment a small moleculecan have a molecular weight of less than about 800 amu. In anotherembodiment a small molecule can have a molecular weight of less thanabout 500 amu. In certain embodiments, the therapeutic agent is asteroid (e.g., dexamethasone). In certain embodiments the therapeuticagent is a NSAID (e.g., a NSAID described herein), which may be the sameor different from the NSAID administered via injection.

In certain embodiments, the therapeutic agent is a nucleic acid. Incertain embodiments, the nucleic acid is mRNA. In certain embodiments,the nucleic acid is an antisense nucleic acid (e.g., siRNA or shRNA)capable of inhibiting transcription or translation of the correspondingmessenger RNA (mRNA). In certain embodiments, the nucleic acid is siRNA.

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, as longerdouble-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. In some embodiments, siRNAmay be produced enzymatically or by partial/total organic synthesis, andmodified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In certain instances, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,e.g., the chemical synthesis methods as described in Verma and Eckstein(1998) or as described herein.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods include Sambrook et al.,Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, GeneTransfer and Expression: A Laboratory Manual (1990); and CurrentProtocols in Molecular Biology (Ausubel et al., eds., 1994). Thedisclosures of these references are herein incorporated by reference intheir entirety for all purposes.

Typically, siRNA are chemically synthesized. The oligonucleotides thatcomprise the siRNA molecules can be synthesized using any of a varietyof techniques known in the art, such as those described in Usman et al.,J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res.,18:5433 (1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995);and Wincott et al., Methods Mol. Bio., 74:59 (1997). The synthesis ofoligonucleotides makes use of common nucleic acid protecting andcoupling groups, such as dimethoxytrityl at the 5′-end andphosphoramidites at the 3′-end. As a non-limiting example, small scalesyntheses can be conducted on an Applied Biosystems synthesizer using a0.2 μmol scale protocol. Alternatively, syntheses at the 0.2 μmol scalecan be performed on a 96-well plate synthesizer from Protogene (PaloAlto, Calif.). However, a larger or smaller scale of synthesis is alsowithin the scope. Suitable reagents for oligonucleotide synthesis,methods for RNA deprotection, and methods for RNA purification are knownto those of skill in the art.

siRNA molecules can be assembled from two distinct oligonucleotides,wherein one oligonucleotide comprises the sense strand and the othercomprises the antisense strand of the siRNA. For example, each strandcan be synthesized separately and joined together by hybridization orligation following synthesis and/or deprotection.

In certain embodiments, the therapeutic agent is selected from the groupconsisting of:

-   -   a) reverse transcriptase inhibitors;    -   b) capsid inhibitors;    -   c) cccDNA formation inhibitors;    -   d) sAg secretion inhibitors;    -   e) oligomeric nucleotides targeted to the Hepatitis B genome;        and    -   f) immunostimulators.        Reverse Transcriptase Inhibitors

In certain embodiments, the reverse transcriptase inhibitor is anucleoside analog.

In certain embodiments, the reverse transcriptase inhibitor is anucleoside analog reverse-transcriptase inhibitor (NARTI or NRTI).

In certain embodiments, the reverse transcriptase inhibitor is anucleotide analog reverse-transcriptase inhibitor (NtARTI or NtRTI).

The term reverse transcriptase inhibitor includes, but is not limitedto: entecavir, clevudine, telbivudine, lamivudine, adefovir, tenofovir,tenofovir disoproxil, tenofovir alafenamide, adefovir dipovoxil,(1R,2R,3R,5R)-3-(6-amino-9H-9-purinyl)-2-fluoro-5-(hydroxymethyl)-4-methylenecyclopentan-1-ol(described in U.S. Pat. No. 8,816,074), emtricitabine, abacavir,elvucitabine, ganciclovir, lobucavir, famciclovir, penciclovir, andamdoxovir.

The term reverse transcriptase inhibitor includes, but is not limitedto, entecavir, lamivudine, and(1R,2R,3R,5R)-3-(6-amino-9H-9-purinyl)-2-fluoro-5-(hydroxymethyl)-4-methylenecyclopentan-1-ol.

The term reverse transcriptase inhibitor includes, but is not limited toa covalently bound phosphoramidate or phosphonamidate moiety of theabove-mentioned reverse transcriptase inhibitors, or as described in,for example, U.S. Pat. No. 8,816,074, US 2011/0245484 A1, and US2008/0286230A1.

The term reverse transcriptase inhibitor includes, but is not limitedto, nucleotide analogs that comprise a phosphoramidate moiety, such as,methyl((((1R,3R,4R,5R)-3-(6-amino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-(Dor L)-alaninate and methyl((((1R,2R,3R,4R)-3-fluoro-2-hydroxy-5-methylene-4-(6-oxo-1,6-dihydro-9H-purin-9-yl)cyclopentyl)methoxy)(phenoxy)phosphoryl)-(Dor L)-alaninate. Also included are the individual diastereomers thereof,which includes, for example, methyl((R)-(((1R,3R,4R,5R)-3-(6-amino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-(Dor L)-alaninate and methyl((S)-(((1R,3R,4R,5R)-3-(6-amino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-(Dor L)-alaninate.

The term reverse transcriptase inhibitor includes, but is not limited toa phosphonamidate moiety, such as, tenofovir alafenamide, as well asthose described in US 2008/0286230 A1. Methods for preparingstereoselective phosphoramidate or phosphonamidate containing activesare described in, for example, U.S. Pat. No. 8,816,074, as well as US2011/0245484 A1 and US 2008/0286230 A1.

Capsid Inhibitors

As described herein the term “capsid inhibitor” includes compounds thatare capable of inhibiting the expression and/or function of a capsidprotein either directly or indirectly. For example, a capsid inhibitormay include, but is not limited to, any compound that inhibits capsidassembly, induces formation of non-capsid polymers, promotes excesscapsid assembly or misdirected capsid assembly, affects capsidstabilization, and/or inhibits encapsidation of RNA. Capsid inhibitorsalso include any compound that inhibits capsid function in a downstreamevent(s) within the replication process (e.g., viral DNA synthesis,transport of relaxed circular DNA (rcDNA) into the nucleus, covalentlyclosed circular DNA (cccDNA) formation, virus maturation, budding and/orrelease, and the like). For example, in certain embodiments, theinhibitor detectably inhibits the expression level or biologicalactivity of the capsid protein as measured, e.g., using an assaydescribed herein. In certain embodiments, the inhibitor inhibits thelevel of rcDNA and downstream products of viral life cycle by at least5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least90%.

The term capsid inhibitor includes compounds described in InternationalPatent Applications Publication Numbers WO2013006394, WO2014106019, andWO2014089296, including the following compounds:

The term capsid inhibitor also includes the compounds Bay-41-4109 (seeInternational Patent Application Publication Number WO/2013/144129),AT-61 (see International Patent Application Publication NumberWO/1998/33501; and King, R W, et al., Antimicrob Agents Chemother, 1998,42, 12, 3179-3186), DVR-O1 and DVR-23 (see International PatentApplication Publication Number WO 2013/006394; and Campagna, M R, etal., J. of Virology, 2013, 87, 12, 6931, and pharmaceutically acceptablesalts thereof:

cccDNA Formation Inhibitors

Covalently closed circular DNA (cccDNA) is generated in the cell nucleusfrom viral rcDNA and serves as the transcription template for viralmRNAs. As described herein, the term “cccDNA formation inhibitor”includes compounds that are capable of inhibiting the formation and/orstability of cccDNA either directly or indirectly. For example, a cccDNAformation inhibitor may include, but is not limited to, any compoundthat inhibits capsid disassembly, rcDNA entry into the nucleus, and/orthe conversion of rcDNA into cccDNA. For example, in certainembodiments, the inhibitor detectably inhibits the formation and/orstability of the cccDNA as measured, e.g., using an assay describedherein. In certain embodiments, the inhibitor inhibits the formationand/or stability of cccDNA by at least 5%, at least 10%, at least 20%,at least 50%, at least 75%, or at least 90%.

The term cccDNA formation inhibitor includes compounds described inInternational Patent Application Publication Number WO2013130703,including the following compounds:

The term cccDNA formation inhibitor includes, but is not limited tothose generally and specifically described in United States PatentApplication Publication Number US 2015/0038515 A1. The term cccDNAformation inhibitor includes, but is not limited to,1-(phenylsulfonyl)-N-(pyridin-4-ylmethyl)-1H-indole-2-carboxamide;1-Benzenesulfonyl-pyrroli dine-2-carboxylic acid(pyridin-4-ylmethyl)-amide;2-(2-chloro-N-(2-chloro-5-(trifluoromethyl)phenyl)-4-(trifluoromethyl)phenylsulfonamido)-N-(pyridin-4-ylmethyl)acetamide;2-(4-chloro-N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-N-(pyridin-4-ylmethyl)acetamide;2-(N-(2-chloro-5-(trifluoromethyl)phenyl)-4-(trifluoromethyl)phenylsulfonamido)-N-(pyridin-4-ylmethyl)acetamide;2-(N-(2-chloro-5-(trifluoromethyl)phenyl)-4-methoxyphenylsulfonamido)-N-(pyridin-4-ylmethyl)acetamide;2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-N-((1-methylpiperidin-4-yl)methyl)acetamide;2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-N-(piperidin-4-ylmethyl)acetamide;2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-N-(pyridin-4-ylmethyl)propanamide;2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-N-(pyridin-3-ylmethyl)acetamide;2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-N-(pyrimidin-5-ylmethyl)acetamide;2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-N-(pyrimidin-4-ylmethyl)acetamide;2-(N-(5-chloro-2-fluorophenyl)phenylsulfonamido)-N-(pyridin-4-ylmethyl)acetamide;2-[(2-chloro-5-trifluoromethyl-phenyl)-(4-fluoro-benzenesulfonyl)-amino]-N-pyridin-4-ylmethyl-acetamide;2-[(2-chloro-5-trifluoromethyl-phenyl)-(toluene-4-sulfonyl)-amino]-N-pyridin-4-ylmethyl-acetamide;2-[benzenesulfonyl-(2-bromo-5-trifluoromethyl-phenyl)-amino]-N-pyridin-4-ylmethyl-acetamide;2-[benzenesulfonyl-(2-chloro-5-trifluoromethyl-phenyl)-amino]-N-(2-methyl-benzothiazol-5-yl)-acetamide;2-[benzenesulfonyl-(2-chloro-5-trifluoromethyl-phenyl)-amino]-N-[4-(4-methyl-piperazin-1-yl)-benzyl]-acetamide;2-[benzenesulfonyl-(2-chloro-5-trifluoromethyl-phenyl)-amino]-N-[3-(4-methyl-piperazin-1-yl)-benzyl]-acetamide;2-[benzenesulfonyl-(2-chloro-5-trifluoromethyl-phenyl)-amino]-N-benzyl-acetamide;2-[benzenesulfonyl-(2-chloro-5-trifluoromethyl-phenyl)-amino]-N-pyridin-4-ylmethyl-acetamide;2-[benzenesulfonyl-(2-chloro-5-trifluoromethyl-phenyl)-amino]-N-pyridin-4-ylmethyl-propionamide;2-[benzenesulfonyl-(2-fluoro-5-trifluoromethyl-phenyl)-amino]-N-pyridin-4-ylmethyl-acetamide;4 (N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-N-(pyridin-4-yl-methyl)butanamide;4-((2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-acetamido)-methyl)-1,1-dimethylpiperidin-1-iumchloride;4-(benzyl-methyl-sulfamoyl)-N-(2-chloro-5-trifluoromethyl-phenyl)-benzamide;4-(benzyl-methyl-sulfamoyl)-N-(2-methyl-1H-indol-5-yl)-benzamide;4-(benzyl-methyl-sulfamoyl)-N-(2-methyl-1H-indol-5-yl)-benzamide;4-(benzyl-methyl-sulfamoyl)-N-(2-methyl-benzothiazol-5-yl)-benzamide;4-(benzyl-methyl-sulfamoyl)-N-(2-methyl-benzothiazol-6-yl)-benzamide;4-(benzyl-methyl-sulfamoyl)-N-(2-methyl-benzothiazol-6-yl)-benzamide;4-(benzyl-methyl-sulfamoyl)-N-pyridin-4-ylmethyl-benzamide;N-(2-aminoethyl)-2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-acetamide;N-(2-chloro-5-(trifluoromethyl)phenyl)-N-(2-(3,4-dihydro-2,6-naphthyridin-2(1H)-yl)-2-oxoethyl)benzenesulfonamide;N-benzothiazol-6-yl-4-(benzyl-methyl-sulfamoyl)-benzamide;N-benzothiazol-6-yl-4-(benzyl-methyl-sulfamoyl)-benzamide; tert-butyl(2-(2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)acetamido)-ethyl)carbamate;and tert-butyl4-((2-(N-(2-chloro-5-(trifluoromethyl)phenyl)phenylsulfonamido)-acetamido)-methyl)piperidine-1-carboxylate,and optionally, combinations thereof.

sAg Secretion Inhibitors

As described herein the term “sAg secretion inhibitor” includescompounds that are capable of inhibiting, either directly or indirectly,the secretion of sAg (S, M and/or L surface antigens) bearing subviralparticles and/or DNA containing viral particles from HBV-infected cells.For example, in certain embodiments, the inhibitor detectably inhibitsthe secretion of sAg as measured, e.g., using assays known in the art ordescribed herein, e.g., ELISA assay or by Western Blot. In certainembodiments, the inhibitor inhibits the secretion of sAg by at least 5%,at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.In certain embodiments, the inhibitor reduces serum levels of sAg in apatient by at least 5%, at least 10%, at least 20%, at least 50%, atleast 75%, or at least 90%.

The term sAg secretion inhibitor includes compounds described in U.S.Pat. No. 8,921,381, as well as compounds described in United StatesPatent Application Publication Numbers 2015/0087659 and 2013/0303552.For example, the term includes the compounds PBHBV-001 and PBHBV-2-15,and pharmaceutically acceptable salts thereof:

Immunostimulators

The term “immunostimulator” includes compounds that are capable ofmodulating an immune response (e.g., stimulate an immune response (e.g.,an adjuvant)). The term immunostimulators includespolyinosinic:polycytidylic acid (poly I:C) and interferons.

The term immunostimulators includes agonists of stimulator of IFN genes(STING) and interleukins. The term also includes HBsAg releaseinhibitors, TLR-7 agonists (GS-9620, RG-7795), T-cell stimulators(GS-4774), RIG-1 inhibitors (SB-9200), and SMAC-mimetics (Birinapant).

Oligomeric Nucleotides

The term oligomeric nucleotide targeted to the Hepatitis B genomeincludes Arrowhead-ARC-520 (see U.S. Pat. No. 8,809,293; and Wooddell CI, et al., Molecular Therapy, 2013, 21, 5, 973-985).

The oligomeric nucleotides can be designed to target one or more genesand/or transcripts of the HBV genome.

The term oligomeric nucleotide targeted to the Hepatitis B genome alsoincludes isolated, double stranded, siRNA molecules, that each include asense strand and an antisense strand that is hybridized to the sensestrand. The siRNA target one or more genes and/or transcripts of the HBVgenome.

Carrier Systems Containing a Therapeutic Agent

Lipid Particles

In certain embodiments of the invention the at least one lipidformulated therapeutic agent is formulated in a lipid nanoparticle (LNP)comprising the at least one therapeutic agent, a cationic lipid and anon-cationic lipid. In certain embodiments, the LNP further comprises aconjugated lipid that inhibits aggregation of particles. In oneembodiment, the lipid particles can comprise one or more therapeuticagents (e.g., a nucleic acid, such as a siRNA or mRNA), a cationiclipid, a non-cationic lipid, and a conjugated lipid that inhibitsaggregation of particles. In some embodiments, the therapeutic agent isfully encapsulated within the lipid portion of the lipid particle. Forexample, in certain embodiments, the therapeutic agent is a nucleic acidthat is fully encapsulated within the lipid portion of the lipidparticle such that the nucleic acid in the lipid particle is resistantin aqueous solution to nuclease degradation. In other embodiments, thelipid particles described herein are substantially non-toxic to mammalssuch as humans. The lipid particles typically have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, or from about 70 to about 90 nm. In certainembodiments, the lipid particles have a median diameter of from about 30nm to about 150 nm. The lipid particles also typically have alipid:therapeutic agent ratio (e.g., a lipid:nucleic acid ratio)(mass/mass ratio) of from about 1:1 to about 100:1, from about 1:1 toabout 50:1, from about 2:1 to about 25:1, from about 3:1 to about 20:1,from about 5:1 to about 15:1, or from about 5:1 to about 10:1. Incertain embodiments, the therapeutic agent-lipid particle has alipid:therapeutic agent mass ratio of from about 5:1 to about 15:1. Incertain embodiments, the therapeutic agent is a nucleic acid and thenucleic acid-lipid particle has a lipid:nucleic acid (e.g., siRNA) massratio of from about 5:1 to about 15:1.

In certain embodiments, the therapeutic agent is a nucleic acid.Accordingly, certain embodiments of the invention include serum-stablenucleic acid-lipid particles which comprise one or more siRNA or mRNAmolecules, a cationic lipid (e.g., one or more cationic lipids ofFormula I-III or salts thereof as set forth herein), a non-cationiclipid (e.g., mixtures of one or more phospholipids and cholesterol), anda conjugated lipid that inhibits aggregation of the particles (e.g., oneor more PEG-lipid conjugates). The lipid particle may comprise at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more siRNA molecules that target agene of interest (e.g., a HBV gene) or mRNA molecules. Nucleicacid-lipid particles and their method of preparation are described in,e.g., U.S. Pat. Nos. 5,753,613; 5,785,992; 5,705,385; 5,976,567;5,981,501; 6,110,745; and 6,320,017; and PCT Publication No. WO96/40964, the disclosures of which are each herein incorporated byreference in their entirety for all purposes.

In the nucleic acid-lipid particles, the one or more nucleic acidmolecules (e.g., an siRNA molecule or mRNA molecule) may be fullyencapsulated within the lipid portion of the particle, therebyprotecting the siRNA from nuclease degradation. In certain instances,the nucleic acid in the nucleic acid-lipid particle is not substantiallydegraded after exposure of the particle to a nuclease at 37° C. for atleast about 20, 30, 45, or 60 minutes. In certain other instances, thenucleic acid in the nucleic acid-lipid particle is not substantiallydegraded after incubation of the particle in serum at 37° C. for atleast about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. Inother embodiments, the nucleic acid is complexed with the lipid portionof the particle. One of the benefits of the formulations is that thenucleic acid-lipid particle compositions are substantially non-toxic tomammals such as humans.

The term “fully encapsulated” indicates that the nucleic acid (e.g., asiRNA molecule or mRNA molecule) in the nucleic acid-lipid particle isnot significantly degraded after exposure to serum or a nuclease assaythat would significantly degrade free DNA or RNA. In a fullyencapsulated system, preferably less than about 25% of the nucleic acidin the particle is degraded in a treatment that would normally degrade100% of free nucleic acid, more preferably less than about 10%, and mostpreferably less than about 5% of the nucleic acid in the particle isdegraded. “Fully encapsulated” also indicates that the nucleicacid-lipid particles are serum-stable, that is, that they do not rapidlydecompose into their component parts upon in vivo administration.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Specific dyes such as OliGreen® and RiboGreen® (Invitrogen Corp.;Carlsbad, Calif.) are available for the quantitative determination ofplasmid DNA, single-stranded deoxyribonucleotides, and/or single- ordouble-stranded ribonucleotides. Encapsulation is determined by addingthe dye to a liposomal formulation, measuring the resultingfluorescence, and comparing it to the fluorescence observed uponaddition of a small amount of nonionic detergent. Detergent-mediateddisruption of the liposomal bilayer releases the encapsulated nucleicacid, allowing it to interact with the membrane-impermeable dye. Nucleicacid encapsulation may be calculated as E=(I_(o)−I)/I_(o), where I andI_(o) refer to the fluorescence intensities before and after theaddition of detergent (see, Wheeler et al., Gene Ther., 6:271-281(1999)).

In some instances, the nucleic acid-lipid particle composition comprisesa nucleic acid molecule (e.g., a siRNA molecule or mRNA molecule) thatis fully encapsulated within the lipid portion of the particles, suchthat from about 30% to about 100%, from about 40% to about 100%, fromabout 50% to about 100%, from about 60% to about 100%, from about 70% toabout 100%, from about 80% to about 100%, from about 90% to about 100%,from about 30% to about 95%, from about 40% to about 95%, from about 50%to about 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the particles have the nucleic acid encapsulated therein.

In other instances, the nucleic acid-lipid particle compositioncomprises nucleic acid that is fully encapsulated within the lipidportion of the particles, such that from about 30% to about 100%, fromabout 40% to about 100%, from about 50% to about 100%, from about 60% toabout 100%, from about 70% to about 100%, from about 80% to about 100%,from about 90% to about 100%, from about 30% to about 95%, from about40% to about 95%, from about 50% to about 95%, from about 60% to about95%, from about 70% to about 95%, from about 80% to about 95%, fromabout 85% to about 95%, from about 90% to about 95%, from about 30% toabout 90%, from about 40% to about 90%, from about 50% to about 90%,from about 60% to about 90%, from about 70% to about 90%, from about 80%to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%(or any fraction thereof or range therein) of the input nucleic acid isencapsulated in the particles.

Depending on the intended use of the lipid particles, the proportions ofthe components can be varied and the delivery efficiency of a particularformulation can be measured using, e.g., an endosomal release parameter(ERP) assay.

Cationic Lipids

Any of a variety of cationic lipids or salts thereof may be used in thelipid particles either alone or in combination with one or more othercationic lipid species or non-cationic lipid species. The cationiclipids include the (R) and/or (S) enantiomers thereof.

In one aspect, the cationic lipid is a dialkyl lipid. For example,dialkyl lipids may include lipids that comprise two saturated orunsaturated alkyl chains, wherein each of the alkyl chains may besubstituted or unsubstituted. In certain embodiments, each of the twoalkyl chains comprise at least, e.g., 8 carbon atoms, 10 carbon atoms,12 carbon atoms, 14 carbon atoms, 16 carbon atoms, 18 carbon atoms, 20carbon atoms, 22 carbon atoms or 24 carbon atoms.

In one aspect, the cationic lipid is a trialkyl lipid. For example,trialkyl lipids may include lipids that comprise three saturated orunsaturated alkyl chains, wherein each of the alkyl chains may besubstituted or unsubstituted. In certain embodiments, each of the threealkyl chains comprise at least, e.g., 8 carbon atoms, 10 carbon atoms,12 carbon atoms, 14 carbon atoms, 16 carbon atoms, 18 carbon atoms, 20carbon atoms, 22 carbon atoms or 24 carbon atoms.

In one aspect, cationic lipids of Formula I having the followingstructure are useful:

or salts thereof, wherein:

R¹ and R² are either the same or different and are independentlyhydrogen (H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl, or R¹ and R² may join to form an optionally substitutedheterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selectedfrom the group consisting of nitrogen (N), oxygen (O), and mixturesthereof;

R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to provide aquaternary amine; R⁴ and R⁵ are either the same or different and areindependently an optionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl,C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and R⁵comprises at least two sites of unsaturation; and

n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In onepreferred embodiment, R¹ and R² are both methyl groups. In otherpreferred embodiments, n is 1 or 2. In other embodiments, R³ is absentwhen the pH is above the pK_(a) of the cationic lipid and R³ is hydrogenwhen the pH is below the pK_(a) of the cationic lipid such that theamino head group is protonated. In an alternative embodiment, R³ is anoptionally substituted C₁-C₄ alkyl to provide a quaternary amine. Infurther embodiments, R⁴ and R⁵ are independently an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl, wherein at leastone of R⁴ and R⁵ comprises at least two sites of unsaturation.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, anarachidonyl moiety, and a docosahexaenoyl moiety, as well as acylderivatives thereof (e.g., linoleoyl, linolenoyl, γ-linolenoyl, etc.).In some instances, one of R⁴ and R⁵ comprises a branched alkyl group(e.g., a phytanyl moiety) or an acyl derivative thereof (e.g., aphytanoyl moiety). In certain instances, the octadecadienyl moiety is alinoleyl moiety. In certain other instances, the octadecatrienyl moietyis a linolenyl moiety or a γ-linolenyl moiety. In certain embodiments,R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties, or γ-linolenylmoieties. In particular embodiments, the cationic lipid of Formula I is1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA),1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDAP), ormixtures thereof.

In some embodiments, the cationic lipid of Formula I forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula I is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLinDMA and DLenDMA, as well asadditional cationic lipids, is described in U.S. Patent Publication No.20060083780, the disclosure of which is herein incorporated by referencein its entirety for all purposes. The synthesis of cationic lipids suchas C2-DLinDMA and C2-DLinDAP, as well as additional cationic lipids, isdescribed in international patent application number WO2011/000106 thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

In another aspect, cationic lipids of Formula II having the followingstructure (or salts thereof) are useful:

wherein R¹ and R² are either the same or different and are independentlyan optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄alkynyl, or C₁₂-C₂₄ acyl; R³ and R⁴ are either the same or different andare independently an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,or C₂-C₆ alkynyl, or R³ and R⁴ may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent or ishydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; m, n, and pare either the same or different and are independently either 0, 1, or2, with the proviso that m, n, and p are not simultaneously 0; q is 0,1, 2, 3, or 4; and Y and Z are either the same or different and areindependently O, S, or NH. In a preferred embodiment, q is 2.

In some embodiments, the cationic lipid of Formula II is2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2” or “C2K”),2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA;“C₃K”), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA; “C4K”),2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA),2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DO-K-DMA),2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DS-K-DMA),2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride(DLin-K-TMA.Cl),2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane(DLin-K²-DMA), 2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane(D-Lin-K-N-methylpiperzine), or mixtures thereof. In one embodiment thecationic lipid of Formula II is DLin-K-C2-DMA.

In some embodiments, the cationic lipid of Formula II forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula II is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLin-K-DMA, as well asadditional cationic lipids, is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. The synthesis of cationic lipids suchas DLin-K-C2-DMA, DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ,DO-K-DMA, DS-K-DMA, DLin-K-MA, DLin-K-TMA.C1, DLin-K²-DMA, andD-Lin-K-N-methylpiperzine, as well as additional cationic lipids, isdescribed in PCT Application No. PCT/US2009/060251, entitled “ImprovedAmino Lipids and Methods for the Delivery of Nucleic Acids,” filed Oct.9, 2009, the disclosure of which is incorporated herein by reference inits entirety for all purposes.

In a further aspect, cationic lipids of Formula III having the followingstructure are useful:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof, R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either absentor present and when present are either the same or different and areindependently an optionally substituted C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl;and n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, R⁴ and R⁵ are both butyl groups. In yet another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substituted C₂-C₆or C₂-C₄ alkyl or C₂-C₆ or C₂-C₄ alkenyl.

In an alternative embodiment, the cationic lipid of Formula IIIcomprises ester linkages between the amino head group and one or both ofthe alkyl chains. In some embodiments, the cationic lipid of Formula IIIforms a salt (preferably a crystalline salt) with one or more anions. Inone particular embodiment, the cationic lipid of Formula III is theoxalate (e.g., hemioxalate) salt thereof, which is preferably acrystalline salt.

Although each of the alkyl chains in Formula III contains cis doublebonds at positions 6, 9, and 12 (i.e., cis,cis,cis-Δ⁶,Δ⁹,Δ¹²), in analternative embodiment, one, two, or three of these double bonds in oneor both alkyl chains may be in the trans configuration.

In a particular embodiment, the cationic lipid of Formula III has thestructure:

The synthesis of cationic lipids such as γ-DLenDMA (15), as well asadditional cationic lipids, is described in U.S. Provisional ApplicationNo. 61/222,462, entitled “Improved Cationic Lipids and Methods for theDelivery of Nucleic Acids,” filed Jul. 1, 2009, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

The synthesis of cationic lipids such as DLin-M-C3-DMA (“MC3”), as wellas additional cationic lipids (e.g., certain analogs of MC3), isdescribed in U.S. Provisional Application No. 61/185,800, entitled“Novel Lipids and Compositions for the Delivery of Therapeutics,” filedJun. 10, 2009, and U.S. Provisional Application No. 61/287,995, entitled“Methods and Compositions for Delivery of Nucleic Acids,” filed Dec. 18,2009, the disclosures of which are herein incorporated by reference intheir entirety for all purposes.

Examples of other cationic lipids or salts thereof which may be includedin the lipid particles include, but are not limited to, cationic lipidssuch as those described in WO2011/000106, the disclosure of which isherein incorporated by reference in its entirety for all purposes, aswell as cationic lipids such as N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-(5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP),1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl),dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA; also known asDLin-M-K-DMA or DLin-M-DMA), and mixtures thereof. Additional cationiclipids or salts thereof which may be included in the lipid particles aredescribed in U.S. Patent Publication No. 20090023673, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

The synthesis of cationic lipids such as CLinDMA, as well as additionalcationic lipids, is described in U.S. Patent Publication No.20060240554, the disclosure of which is herein incorporated by referencein its entirety for all purposes. The synthesis of cationic lipids suchas DLin-C-DAP, DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLin-2-DMAP,DLinTMA.Cl, DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, and DLin-EG-DMA, as wellas additional cationic lipids, is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. The synthesis of cationic lipids suchas DO-C-DAP, DMDAP, DOTAP.Cl, DLin-M-C2-DMA, as well as additionalcationic lipids, is described in PCT Application No. PCT/US2009/060251,entitled “Improved Amino Lipids and Methods for the Delivery of NucleicAcids,” filed Oct. 9, 2009, the disclosure of which is incorporatedherein by reference in its entirety for all purposes. The synthesis of anumber of other cationic lipids and related analogs has been describedin U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613;and 5,785,992; and PCT Publication No. WO 96/10390, the disclosures ofwhich are each herein incorporated by reference in their entirety forall purposes. Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN® (including DOTMAand DOPE, available from Invitrogen); LIPOFECTAMINE® (including DOSPAand DOPE, available from Invitrogen); and TRANSFECTAM® (including DOGS,available from Promega Corp.).

In some embodiments, the cationic lipid comprises from about 50 mol % toabout 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol% to about 80 mol %, from about 50 mol % to about 75 mol %, from about50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, fromabout 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %,or from about 55 mol % to about 70 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle. In particularembodiments, the cationic lipid comprises about 50 mol %, 51 mol %, 52mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (orany fraction thereof) of the total lipid present in the particle.

In other embodiments, the cationic lipid comprises from about 2 mol % toabout 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol% to about 50 mol %, from about 20 mol % to about 50 mol %, from about20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, orabout 40 mol % (or any fraction thereof or range therein) of the totallipid present in the particle.

Additional percentages and ranges of cationic lipids suitable for use inthe lipid particles are described in PCT Publication No. WO 09/127060,U.S. Published Application No. US 2011/0071208, PCT Publication No.WO2011/000106, and U.S. Published Application No. US 2011/0076335, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

It should be understood that the percentage of cationic lipid present inthe lipid particles is a target amount, and that the actual amount ofcationic lipid present in the formulation may vary, for example, by ±5mol %. For example, in one exemplary lipid particle formulation, thetarget amount of cationic lipid is 57.1 mol %, but the actual amount ofcationic lipid may be ±5 mol %, ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that targetamount, with the balance of the formulation being made up of other lipidcomponents (adding up to 100 mol % of total lipids present in theparticle; however, one skilled in the art will understand that the totalmol % may deviate slightly from 100% due to rounding, for example, 99.9mol % or 100.1 mol %).

Further examples of cationic lipids useful for inclusion in lipidparticles are shown below:

Non-Cationic Lipids

The non-cationic lipids used in the lipid particles can be any of avariety of neutral uncharged, zwitterionic, or anionic lipids capable ofproducing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphati dylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphati dylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5α-cholestanol,5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether,cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polaranalogues such as 5α-cholestane, cholestenone, 5α-cholestanone,5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Inpreferred embodiments, the cholesterol derivative is a polar analoguesuch as cholesteryl-(4′-hydroxy)-butyl ether. The synthesis ofcholesteryl-(2′-hydroxy)-ethyl ether is described in PCT Publication No.WO 09/127060, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

In some embodiments, the non-cationic lipid present in the lipidparticles comprises or consists of a mixture of one or morephospholipids and cholesterol or a derivative thereof. In otherembodiments, the non-cationic lipid present in the lipid particlescomprises or consists of one or more phospholipids, e.g., acholesterol-free lipid particle formulation. In yet other embodiments,the non-cationic lipid present in the lipid particles comprises orconsists of cholesterol or a derivative thereof, e.g., aphospholipid-free lipid particle formulation.

Other examples of non-cationic lipids suitable for use includenonphosphorous containing lipids such as, e.g., stearylamine,dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate,hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers,triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylatedfatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide,sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 10 mol% to about 60 mol %, from about 20 mol % to about 55 mol %, from about20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %,from about 30 mol % to about 50 mol %, from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 35 mol % toabout 45 mol %, from about 37 mol % to about 45 mol %, or about 35 mol%, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %,43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60mol % of the total lipid present in the particle.

In some embodiments, the phospholipid component in the mixture maycomprise from about 2 mol % to about 20 mol %, from about 2 mol % toabout 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol %to about 15 mol %, or from about 4 mol % to about 10 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. In an certain embodiments, the phospholipid component in themixture comprises from about 5 mol % to about 17 mol %, from about 7 mol% to about 17 mol %, from about 7 mol % to about 15 mol %, from about 8mol % to about 15 mol %, or about 8 mol %, 9 mol %, 10 mol %, 11 mol %,12 mol %, 13 mol %, 14 mol %, or 15 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle. As anon-limiting example, a lipid particle formulation comprising a mixtureof phospholipid and cholesterol may comprise a phospholipid such as DPPCor DSPC at about 7 mol % (or any fraction thereof), e.g., in a mixturewith cholesterol or a cholesterol derivative at about 34 mol % (or anyfraction thereof) of the total lipid present in the particle. As anothernon-limiting example, a lipid particle formulation comprising a mixtureof phospholipid and cholesterol may comprise a phospholipid such as DPPCor DSPC at about 7 mol % (or any fraction thereof), e.g., in a mixturewith cholesterol or a cholesterol derivative at about 32 mol % (or anyfraction thereof) of the total lipid present in the particle.

By way of further example, a lipid formulation useful has a lipid totherapeutic agent (e.g., nucleic acid) ratio of about 10:1 (e.g., alipid:therapeutic agent ratio of from 9.5:1 to 11:1, or from 9.9:1 to11:1, or from 10:1 to 10.9:1). In certain other embodiments, a lipidformulation useful has a lipid to therapeutic agent (e.g., nucleic acid)ratio of about 9:1 (e.g., a lipid:therapeutic agent ratio of from 8.5:1to 10:1, or from 8.9:1 to 10:1, or from 9:1 to 9.9:1, including 9.1:1,9.2:1, 9.3:1, 9.4:1, 9.5:1, 9.6:1, 9.7:1, and 9.8:1).

In other embodiments, the cholesterol component in the mixture maycomprise from about 25 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol% to about 40 mol %, from about 27 mol % to about 37 mol %, from about25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the cholesterol component inthe mixture comprises from about 25 mol % to about 35 mol %, from about27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, fromabout 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %,from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In embodiments where the lipid particles are phospholipid-free, thecholesterol or derivative thereof may comprise up to about 25 mol %, 30mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % ofthe total lipid present in the particle.

In some embodiments, the cholesterol or derivative thereof in thephospholipid-free lipid particle formulation may comprise from about 25mol % to about 45 mol %, from about 25 mol % to about 40 mol %, fromabout 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %,from about 31 mol % to about 39 mol %, from about 32 mol % to about 38mol %, from about 33 mol % to about 37 mol %, from about 35 mol % toabout 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol% to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a lipid particle formulation maycomprise cholesterol at about 37 mol % (or any fraction thereof) of thetotal lipid present in the particle. As another non-limiting example, alipid particle formulation may comprise cholesterol at about 35 mol %(or any fraction thereof) of the total lipid present in the particle.

In other embodiments, the non-cationic lipid comprises from about 5 mol% to about 90 mol %, from about 10 mol % to about 85 mol %, from about20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), orabout 60 mol % (e.g., phospholipid and cholesterol or derivativethereof) (or any fraction thereof or range therein) of the total lipidpresent in the particle.

Additional percentages and ranges of non-cationic lipids suitable foruse in the lipid particles are described in PCT Publication No. WO09/127060, U.S. Published Application No. US 2011/0071208, PCTPublication No. WO2011/000106, and U.S. Published Application No. US2011/0076335, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

It should be understood that the percentage of non-cationic lipidpresent in the lipid particles is a target amount, and that the actualamount of non-cationic lipid present in the formulation may vary, forexample, by ±5 mol %, ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol%, ±0.5 mol %, ±0.25 mol %, or 0.1 mol %.

Lipid Conjugates

In addition to cationic and non-cationic lipids, the lipid particles mayfurther comprise a lipid conjugate. The conjugated lipid is useful inthat it prevents the aggregation of particles. Suitable conjugatedlipids include, but are not limited to, PEG-lipid conjugates, POZ-lipidconjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates(CPLs), and mixtures thereof. In certain embodiments, the particlescomprise either a PEG-lipid conjugate or an ATTA-lipid conjugatetogether with a CPL.

In a preferred embodiment, the lipid conjugate is a PEG-lipid conjugate.Examples of PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes.

Additional PEG-lipids suitable for use include, without limitation,mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). Thesynthesis of PEG-C-DOMG is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. Yet additional suitable PEG-lipidconjugates include, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, but are not limited to, thefollowing: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S—NHS, HO-PEG-NH₂, etc.).Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing thePEG-lipid conjugates. The disclosures of these patents are hereinincorporated by reference in their entirety for all purposes. Inaddition, monomethoxypolyethyleneglycol-acetic acid (MePEG-CH₂COOH) isparticularly useful for preparing PEG-lipid conjugates including, e.g.,PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)-), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O-(O)POH-O-), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skill in the art.Phosphatidyl-ethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

The term “ATTA” or “polyamide” includes, without limitation, compoundsdescribed in U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. These compounds include a compound having the formula:

wherein R is a member selected from the group consisting of hydrogen,alkyl and acyl; R¹ is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R¹ and the nitrogen to whichthey are bound form an azido moiety; R² is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be.

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, R¹ and R², both of which have independently between 2 and30 carbons bonded to the 1- and 2-position of glycerol by esterlinkages. The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited to,lauroyl (C₁₂), myristoyl (C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈), andicosoyl (C₂₀). In preferred embodiments, R¹ and R² are the same, i.e.,R¹ and R² are both myristoyl (i.e., dimyristoyl), R¹ and R² are bothstearoyl (i.e., distearoyl), etc. Diacylglycerols have the followinggeneral formula:

The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkylchains, R¹ and R², both of which have independently between 2 and 30carbons. The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate havingthe following formula:

wherein R¹ and R² are independently selected and are long-chain alkylgroups having from about 10 to about 22 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester containing linker moiety or anester containing linker moiety as described above. The long-chain alkylgroups can be saturated or unsaturated. Suitable alkyl groups include,but are not limited to, decyl (C₁₀), lauryl (C₁₂), myristyl (C₁₄),palmityl (C₁₆), stearyl (C₁₈), and icosyl (C₂₀). In preferredembodiments, R¹ and R² are the same, i.e., R¹ and R² are both myristyl(i.e., dimyristyl), R¹ and R² are both stearyl (i.e., distearyl), etc.

In Formula VII above, the PEG has an average molecular weight rangingfrom about 550 daltons to about 10,000 daltons. In certain instances,the PEG has an average molecular weight of from about 750 daltons toabout 5,000 daltons (e.g., from about 1,000 daltons to about 5,000daltons, from about 1,500 daltons to about 3,000 daltons, from about 750daltons to about 3,000 daltons, from about 750 daltons to about 2,000daltons, etc.). In preferred embodiments, the PEG has an averagemolecular weight of about 2,000 daltons or about 750 daltons. The PEGcan be optionally substituted with alkyl, alkoxy, acyl, or aryl groups.In certain embodiments, the terminal hydroxyl group is substituted witha methoxy or methyl group.

In a preferred embodiment, “L” is a non-ester containing linker moiety.Suitable non-ester containing linkers include, but are not limited to,an amido linker moiety, an amino linker moiety, a carbonyl linkermoiety, a carbamate linker moiety, a urea linker moiety, an ether linkermoiety, a disulphide linker moiety, a succinamidyl linker moiety, andcombinations thereof. In a preferred embodiment, the non-estercontaining linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAAconjugate). In another preferred embodiment, the non-ester containinglinker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate).In yet another preferred embodiment, the non-ester containing linkermoiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

In particular embodiments, the PEG-lipid conjugate is selected from:

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate, and urea linkages. Those of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such techniques are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C₁₀)conjugate, a PEG-dilauryloxypropyl (C₁₂) conjugate, aPEG-dimyristyloxypropyl (C₁₄) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, or a PEG-distearyloxypropyl (C₁₈) conjugate. In theseembodiments, the PEG preferably has an average molecular weight of about750 or about 2,000 daltons. In one particularly preferred embodiment,the PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the “2000”denotes the average molecular weight of the PEG, the “C” denotes acarbamate linker moiety, and the “DMA” denotes dimyristyloxypropyl. Inanother particularly preferred embodiment, the PEG-lipid conjugatecomprises PEG750-C-DMA, wherein the “750” denotes the average molecularweight of the PEG, the “C” denotes a carbamate linker moiety, and the“DMA” denotes dimyristyloxypropyl. In particular embodiments, theterminal hydroxyl group of the PEG is substituted with a methyl group.Those of skill in the art will readily appreciate that otherdialkyloxypropyls can be used in the PEG-DAA conjugates.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the lipid particles can furthercomprise cationic poly(ethylene glycol) (PEG) lipids or CPLs (see, e.g.,Chen et al., Bioconj. Chem., 11:433-437 (2000); U.S. Pat. No. 6,852,334;PCT Publication No. WO 00/62813, the disclosures of which are hereinincorporated by reference in their entirety for all purposes).

Suitable CPLs include compounds of Formula VIII:A-W-Y  (VIII),wherein A, W, and Y are as described below.

With reference to Formula VIII, “A” is a lipid moiety such as anamphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts asa lipid anchor. Suitable lipid examples include, but are not limited to,diacylglycerolyls, dialkylglycerolyls, N-N-dialkylaminos,1,2-diacyloxy-3-aminopropanes, and 1,2-dialkyl-3-aminopropanes.

“W” is a polymer or an oligomer such as a hydrophilic polymer oroligomer. Preferably, the hydrophilic polymer is a biocompatable polymerthat is nonimmunogenic or possesses low inherent immunogenicity.Alternatively, the hydrophilic polymer can be weakly antigenic if usedwith appropriate adjuvants. Suitable nonimmunogenic polymers include,but are not limited to, PEG, polyamides, polylactic acid, polyglycolicacid, polylactic acid/polyglycolic acid copolymers, and combinationsthereof. In a preferred embodiment, the polymer has a molecular weightof from about 250 to about 7,000 daltons.

“Y” is a polycationic moiety. The term polycationic moiety refers to acompound, derivative, or functional group having a positive charge,preferably at least 2 positive charges at a selected pH, preferablyphysiological pH. Suitable polycationic moieties include basic aminoacids and their derivatives such as arginine, asparagine, glutamine,lysine, and histidine; spermine; spermidine; cationic dendrimers;polyamines; polyamine sugars; and amino polysaccharides. Thepolycationic moieties can be linear, such as linear tetralysine,branched or dendrimeric in structure. Polycationic moieties have betweenabout 2 to about 15 positive charges, preferably between about 2 toabout 12 positive charges, and more preferably between about 2 to about8 positive charges at selected pH values. The selection of whichpolycationic moiety to employ may be determined by the type of particleapplication which is desired.

The charges on the polycationic moieties can be either distributedaround the entire particle moiety, or alternatively, they can be adiscrete concentration of charge density in one particular area of theparticle moiety e.g., a charge spike. If the charge density isdistributed on the particle, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed.

The lipid “A” and the nonimmunogenic polymer “W” can be attached byvarious methods and preferably by covalent attachment. Methods known tothose of skill in the art can be used for the covalent attachment of “A”and “W.” Suitable linkages include, but are not limited to, amide,amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. Itwill be apparent to those skilled in the art that “A” and “W” must havecomplementary functional groups to effectuate the linkage. The reactionof these two groups, one on the lipid and the other on the polymer, willprovide the desired linkage. For example, when the lipid is adiacylglycerol and the terminal hydroxyl is activated, for instance withNHS and DCC, to form an active ester, and is then reacted with a polymerwhich contains an amino group, such as with a polyamide (see, e.g., U.S.Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are hereinincorporated by reference in their entirety for all purposes), an amidebond will form between the two groups.

In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand or a chelating moiety forcomplexing calcium. Preferably, after the ligand is attached, thecationic moiety maintains a positive charge. In certain instances, theligand that is attached has a positive charge. Suitable ligands include,but are not limited to, a compound or device with a reactive functionalgroup and include lipids, amphipathic lipids, carrier compounds,bioaffinity compounds, biomaterials, biopolymers, biomedical devices,analytically detectable compounds, therapeutically active compounds,enzymes, peptides, proteins, antibodies, immune stimulators,radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0.1 mol % to about 3 mol %, from about 0.5 mol % to about 3mol %, or about 0.6 mol %, 0.7 mol %, 0.8 mol %, 0.9 mol %, 1.0 mol %,1.1 mol %, 1.2 mol %, 1.3 mol %, 1.4 mol %, 1.5 mol %, 1.6 mol %, 1.7mol %, 1.8 mol %, 1.9 mol %, 2.0 mol %, 2.1 mol %, 2.2 mol %, 2.3 mol %,2.4 mol %, 2.5 mol %, 2.6 mol %, 2.7 mol %, 2.8 mol %, 2.9 mol % or 3mol % (or any fraction thereof or range therein) of the total lipidpresent in the particle.

In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % toabout 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol %to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5mol % to about 12 mol %, or about 2 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle.

In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol%, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol% (or any fraction thereof or range therein) of the total lipid presentin the particle.

It should be understood that the percentage of lipid conjugate presentin the lipid particles is a target amount, and that the actual amount oflipid conjugate present in the formulation may vary, for example, by +5mol %, +4 mol %, +3 mol %, +2 mol %, +1 mol %, +0.75 mol %, +0.5 mol %,+0.25 mol %, or ±0.1 mol %.

Additional percentages and ranges of lipid conjugates suitable for usein the lipid particles are described in PCT Publication No. WO09/127060, U.S. Published Application No. US 2011/0071208, PCTPublication No. WO2011/000106, and U.S. Published Application No. US2011/0076335, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

One of ordinary skill in the art will appreciate that the concentrationof the lipid conjugate can be varied depending on the lipid conjugateemployed and the rate at which the lipid particle is to becomefusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid particle and, in turn, the rate at which the lipid particlebecomes fusogenic. For instance, when a PEG-DAA conjugate is used as thelipid conjugate, the rate at which the lipid particle becomes fusogeniccan be varied, for example, by varying the concentration of the lipidconjugate, by varying the molecular weight of the PEG, or by varying thechain length and degree of saturation of the alkyl groups on the PEG-DAAconjugate. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the lipid particle becomes fusogenic. Other methods whichcan be used to control the rate at which the lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure. Also, by controlling the composition and concentrationof the lipid conjugate, one can control the lipid particle size.

Description of Certain Therapeutic Agent-Lipid Particle Embodiments

A therapeutic agent-lipid particle typically may comprise one or moretherapeutic agents, such as one or more nucleic acid molecules (e.g., acocktail), a cationic lipid, and a non-cationic lipid. In certaininstances, the therapeutic agent-lipid particles further comprise aconjugated lipid that inhibits aggregation of particles.

In some embodiments, the therapeutic agent (e.g., a nucleic acidmolecule, such as a siRNA or mRNA) is fully encapsulated in thetherapeutic agent-lipid particle. With respect to formulationscomprising a cocktail of therapeutic agents, the different types ofspecies present in the cocktail (e.g., siRNA compounds with differentsequences) may be co-encapsulated in the same particle, or each type ofspecies present in the cocktail may be encapsulated in a separateparticle. The cocktail may be formulated in the particles describedherein using a mixture of two, three or more individual agents (e.g.,individuals nucleic acid molecules, each having a unique sequence) atidentical, similar, or different concentrations or molar ratios. In oneembodiment, a cocktail of nucleic acid molecules (corresponding to aplurality of nucleic acid molecules with different sequences) isformulated using identical, similar, or different concentrations ormolar ratios of each species, and the different types of molecules areco-encapsulated in the same particle. In another embodiment, each typeof nucleic acid molecule species present in the cocktail is encapsulatedin different particles at identical, similar, or different nucleic acidmolecule concentrations or molar ratios, and the particles thus formed(each containing a different nucleic acid molecule payload) areadministered separately (e.g., at different times in accordance with atherapeutic regimen), or are combined and administered together as asingle unit dose (e.g., with a pharmaceutically acceptable carrier). Theparticles described herein are serum-stable, are resistant to nucleasedegradation, and are substantially non-toxic to mammals such as humans.

The cationic lipid in the therapeutic agent-lipid particles of theinvention may comprise, e.g., one or more cationic lipids of FormulaI-III described herein or any other cationic lipid species. In oneembodiment, cationic lipid is a dialkyl lipid. In another embodiment,the cationic lipid is a trialkyl lipid. In one particular embodiment,the cationic lipid is selected from the group consisting of1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA; Compound(15)), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate(DLin-M-C2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-M-C3-DMA; Compound (7)), salts thereof,and mixtures thereof.

In another particular embodiment, the cationic lipid is selected fromthe group consisting of 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA; Compound(15)),3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine(DLin-MP-DMA; Compound (8)),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) (Compound (7)),(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate (Compound (13)), a salt thereof, or amixture thereof.

In certain embodiments, the cationic lipid comprises from about 48 mol %to about 62 mol % of the total lipid present in the particle.

The non-cationic lipid in the therapeutic agent-lipid particles of thepresent invention may comprise, e.g., one or more anionic lipids and/orneutral lipids. In some embodiments, the non-cationic lipid comprisesone of the following neutral lipid components: (1) a mixture of aphospholipid and cholesterol or a derivative thereof; (2) cholesterol ora derivative thereof; or (3) a phospholipid. In certain preferredembodiments, the phospholipid comprises dipalmitoylphosphatidylcholine(DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof. In apreferred embodiment, the non-cationic lipid is a mixture of DPPC andcholesterol. In a preferred embodiment, the non-cationic lipid is amixture of DSPC and cholesterol.

In certain embodiments, the non-cationic lipid comprises a mixture of aphospholipid and cholesterol or a derivative thereof, wherein thephospholipid comprises from about 7 mol % to about 17 mol % of the totallipid present in the particle and the cholesterol or derivative thereofcomprises from about 25 mol % to about 40 mol % of the total lipidpresent in the particle.

The lipid conjugate in the therapeutic agent-lipid particles of theinvention inhibits aggregation of particles and may comprise, e.g., oneor more of the lipid conjugates described herein. In one particularembodiment, the lipid conjugate comprises a PEG-lipid conjugate.Examples of PEG-lipid conjugates include, but are not limited to,PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof. In certainembodiments, the PEG-lipid conjugate is selected from the groupconsisting of a PEG-diacylglycerol (PEG-DAG) conjugate, aPEG-dialkyloxypropyl (PEG-DAA) conjugate, a PEG-phospholipid conjugate,a PEG-ceramide (PEG-Cer) conjugate, a PEG-dimyristyloxypropyl (PEG-DMA)conjugate and a mixture thereof. In certain embodiments, the PEG-lipidconjugate is a PEG-DAA conjugate. In certain embodiments, the PEG-DAAconjugate in the lipid particle may comprise a PEG-didecyloxypropyl(C₁₀) conjugate, a PEG-dilauryloxypropyl (C₁₂) conjugate, aPEG-dimyristyloxypropyl (C₁₄) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, a PEG-distearyloxypropyl (C₁₈) conjugate, or mixturesthereof. In certain embodiments, wherein the PEG-DAA conjugate is aPEG-dimyristyloxypropyl (C₁₄) conjugate. In another embodiment, thePEG-DAA conjugate is a compound (66) (PEG-C-DMA) conjugate (e.g.,PEG2000-C-DMA). In another embodiment, the lipid conjugate comprises aPOZ-lipid conjugate such as a POZ-DAA conjugate.

In certain embodiments, the conjugated lipid that inhibits aggregationof particles comprises from about 0.5 mol % to about 3 mol % of thetotal lipid present in the particle.

In certain embodiments, the therapeutic agent-lipid particle has a totallipid:therapeutic agent mass ratio of from about 5:1 to about 15:1.

In certain embodiments, the therapeutic agent-lipid particle has amedian diameter of from about 30 nm to about 150 nm.

In certain embodiments, the therapeutic agent-lipid particle has anelectron dense core.

In some embodiments, the present invention provides therapeuticagent-lipid particles comprising: (a) at least one therapeutic agent;(b) one or more cationic lipids or salts thereof comprising from about50 mol % to about 85 mol % of the total lipid present in the particle;(c) one or more non-cationic lipids comprising from about 13 mol % toabout 49.5 mol % of the total lipid present in the particle; and (d) oneor more conjugated lipids that inhibit aggregation of particlescomprising from about 0.5 mol % to about 2 mol % of the total lipidpresent in the particle.

In one aspect of this embodiment, the therapeutic agent-lipid particlecomprises: (a) at least one therapeutic agent; (b) a cationic lipid or asalt thereof comprising from about 52 mol % to about 62 mol % of thetotal lipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 36 mol % toabout 47 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 1 mol % to about 2 mol % ofthe total lipid present in the particle. In one particular embodiment,the formulation is a four-component system comprising about 1.4 mol %PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 57.1 mol % cationiclipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7.1 mol % DPPC (orDSPC), and about 34.3 mol % cholesterol (or derivative thereof).

In another aspect of this embodiment, the therapeutic agent-lipidparticle comprises: (a) at least one therapeutic agent; (b) a cationiclipid or a salt thereof comprising from about 56.5 mol % to about 66.5mol % of the total lipid present in the particle; (c) cholesterol or aderivative thereof comprising from about 31.5 mol % to about 42.5 mol %of the total lipid present in the particle; and (d) a PEG-lipidconjugate comprising from about 1 mol % to about 2 mol % of the totallipid present in the particle. In one particular embodiment, theformulation is a three-component system which is phospholipid-free andcomprises about 1.5 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA),about 61.5 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof,and about 36.9 mol % cholesterol (or derivative thereof).

Additional formulations are described in PCT Publication No. WO09/127060 and published US patent application publication number US2011/0071208 A1, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

In other embodiments, the present invention provides therapeuticagent-lipid particles comprising: (a) at least one therapeutic agent;(b) one or more cationic lipids or salts thereof comprising from about 2mol % to about 50 mol % of the total lipid present in the particle; (c)one or more non-cationic lipids comprising from about 5 mol % to about90 mol % of the total lipid present in the particle; and (d) one or moreconjugated lipids that inhibit aggregation of particles comprising fromabout 0.5 mol % to about 20 mol % of the total lipid present in theparticle.

In one aspect of this embodiment, the therapeutic agent-lipid particlecomprises: (a) at least one therapeutic agent; (b) a cationic lipid or asalt thereof comprising from about 30 mol % to about 50 mol % of thetotal lipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 47 mol % toabout 69 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 1 mol % to about 3 mol % ofthe total lipid present in the particle. In one particular embodiment,the formulation is a four-component system which comprises about 2 mol %PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 40 mol % cationic lipid(e.g., DLin-K-C2-DMA) or a salt thereof, about 10 mol % DPPC (or DSPC),and about 48 mol % cholesterol (or derivative thereof).

In further embodiments, the present invention provides therapeuticagent-lipid particles comprising: (a) at least one therapeutic agent;(b) one or more cationic lipids or salts thereof comprising from about50 mol % to about 65 mol % of the total lipid present in the particle;(c) one or more non-cationic lipids comprising from about 25 mol % toabout 45 mol % of the total lipid present in the particle; and (d) oneor more conjugated lipids that inhibit aggregation of particlescomprising from about 5 mol % to about 10 mol % of the total lipidpresent in the particle.

In one aspect of this embodiment, the therapeutic agent-lipid particlecomprises: (a) at least one therapeutic agent; (b) a cationic lipid or asalt thereof comprising from about 50 mol % to about 60 mol % of thetotal lipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 35 mol % toabout 45 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 5 mol % to about 10 mol % ofthe total lipid present in the particle. In certain instances, thenon-cationic lipid mixture in the formulation comprises: (i) aphospholipid of from about 5 mol % to about 10 mol % of the total lipidpresent in the particle; and (ii) cholesterol or a derivative thereof offrom about 25 mol % to about 35 mol % of the total lipid present in theparticle. In one particular embodiment, the formulation is afour-component system which comprises about 7 mol % PEG-lipid conjugate(e.g., PEG750-C-DMA), about 54 mol % cationic lipid (e.g.,DLin-K-C2-DMA) or a salt thereof, about 7 mol % DPPC (or DSPC), andabout 32 mol % cholesterol (or derivative thereof).

In another aspect of this embodiment, the therapeutic agent-lipidparticle comprises: (a) at least one therapeutic agent; (b) a cationiclipid or a salt thereof comprising from about 55 mol % to about 65 mol %of the total lipid present in the particle; (c) cholesterol or aderivative thereof comprising from about 30 mol % to about 40 mol % ofthe total lipid present in the particle; and (d) a PEG-lipid conjugatecomprising from about 5 mol % to about 10 mol % of the total lipidpresent in the particle. In one particular embodiment, the formulationis a three-component system which is phospholipid-free and comprisesabout 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about 58 mol %cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, and about 35 mol% cholesterol (or derivative thereof).

Additional embodiments of useful formulations are described in publishedUS patent application publication number US 2011/0076335 A1, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

In certain embodiments of the invention, the therapeutic agent-lipidparticle comprises: (a) at least one therapeutic agent; (b) a cationiclipid or a salt thereof comprising from about 48 mol % to about 62 mol %of the total lipid present in the particle; (c) a mixture of aphospholipid and cholesterol or a derivative thereof, wherein thephospholipid comprises about 7 mol % to about 17 mol % of the totallipid present in the particle, and wherein the cholesterol or derivativethereof comprises about 25 mol % to about 40 mol % of the total lipidpresent in the particle; and (d) a PEG-lipid conjugate comprising fromabout 0.5 mol % to about 3.0 mol % of the total lipid present in theparticle. Exemplary lipid formulations A-Z of this aspect of theinvention are included below.

Exemplary lipid formulation A includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.2%), cationic lipid (53.2%), phospholipid (9.3%), cholesterol(36.4%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(1.2%), the cationic lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA) (53.2%), the phospholipid is DPPC (9.3%), and cholesterol ispresent at 36.4%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation A, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation B which includes the following components(wherein the percentage values of the components are mole percent):PEG-lipid (0.8%), cationic lipid (59.7%), phospholipid (14.2%),cholesterol (25.3%), wherein the actual amounts of the lipids presentmay vary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, inone representative embodiment, the PEG-lipid is PEG-C-DOMG (compound(67)) (0.8%), the cationic lipid is1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (59.7%), thephospholipid is DSPC (14.2%), and cholesterol is present at 25.3%,wherein the actual amounts of the lipids present may vary by, e.g., ±5%(or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol%, ±0.25 mol %, or ±0.1 mol %). Thus, certain embodiments of theinvention provide a therapeutic agent-lipid particle based onformulation B, which comprises at least one therapeutic agent describedherein (e.g., a nucleic acid molecule).

Exemplary lipid formulation C includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.9%), cationic lipid (52.5%), phospholipid (14.8%), cholesterol(30.8%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(1.9%), the cationic lipid is1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA; Compound(15)) (52.5%), the phospholipid is DSPC (14.8%), and cholesterol ispresent at 30.8%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation C, which comprises at least one therapeutic agentdescribed herein (e.g., nucleic acid molecule).

Exemplary lipid formulation D includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(0.7%), cationic lipid (60.3%), phospholipid (8.4%), cholesterol(30.5%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(0.7%), the cationic lipid is3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine(DLin-MP-DMA; Compound (8) (60.3%), the phospholipid is DSPC (8.4%), andcholesterol is present at 30.5%, wherein the actual amounts of thelipids present may vary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %).Thus, certain embodiments of the invention provide a therapeuticagent-lipid particle based on formulation D, which comprises at leastone therapeutic agent described herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation E includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.8%), cationic lipid (52.1%), phospholipid (7.5%), cholesterol(38.5%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(1.8%), the cationic lipid is(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) (Compound (7)) (52.1%), the phospholipid isDPPC (7.5%), and cholesterol is present at 38.5%, wherein the actualamounts of the lipids present may vary by, e.g., ±5% (or e.g., ±4 mol %,±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or±0.1 mol %). Thus, certain embodiments of the invention provide atherapeutic agent-lipid particle based on formulation E, which comprisesat least one therapeutic agent described herein (e.g., a nucleic acidmolecule).

Exemplary formulation F includes the following components (wherein thepercentage values of the components are mole percent): PEG-lipid (0.9%),cationic lipid (57.1%), phospholipid (8.1%), cholesterol (33.8%),wherein the actual amounts of the lipids present may vary by, e.g., ±5%(or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol%, ±0.25 mol %, or ±0.1 mol %). For example, in one representativeembodiment, the PEG-lipid is PEG-C-DOMG (compound (67)) (0.9%), thecationic lipid is 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA; Compound(15)) (57.1%), the phospholipid is DSPC (8.1%), and cholesterol ispresent at 33.8%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation F, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation G includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.7%), cationic lipid (61.6%), phospholipid (11.2%), cholesterol(25.5%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(1.7%), the cationic lipid is1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA; Compound(15)) (61.6%), the phospholipid is DPPC (11.2%), and cholesterol ispresent at 25.5%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation G, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation H includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.1%), cationic lipid (55.0%), phospholipid (11.0%), cholesterol(33.0%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(1.1%), the cationic lipid is(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate (Compound (13)) (55.0%), the phospholipid isDSPC (11.0%), and cholesterol is present at 33.0%, wherein the actualamounts of the lipids present may vary by, e.g., ±5% (or e.g., ±4 mol %,±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or±0.1 mol %). Thus, certain embodiments of the invention provide atherapeutic agent-lipid particle based on formulation H, which comprisesat least one therapeutic agent described herein (e.g., a nucleic acidmolecule).

Exemplary lipid formulation I includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.6%), cationic lipid (53.1%), phospholipid (9.4%), cholesterol(35.0%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(2.6%), the cationic lipid is(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate (Compound (13)) (53.1%), the phospholipid isDSPC (9.4%), and cholesterol is present at 35.0%, wherein the actualamounts of the lipids present may vary by, e.g., ±5% (or e.g., ±4 mol %,±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or±0.1 mol %). Thus, certain embodiments of the invention provide atherapeutic agent-lipid particle based on formulation I, which comprisesat least one therapeutic agent described herein (e.g., a nucleic acidmolecule).

Exemplary lipid formulation J includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(0.6%), cationic lipid (59.4%), phospholipid (10.2%), cholesterol(29.8%), wherein the actual amounts of the lipids present may vary byby, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(0.6%), the cationic lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA) (59.4%), the phospholipid is DPPC (10.2%), and cholesterol ispresent at 29.8%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation J, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation K includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(0.5%), cationic lipid (56.7%), phospholipid (13.1%), cholesterol(29.7%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(0.5%), the cationic lipid is(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) (Compound (7)) (56.7%), the phospholipid isDSPC (13.1%), and cholesterol is present at 29.7%, wherein the actualamounts of the lipids present may vary by, e.g., ±5% (or e.g., ±4 mol %,±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or±0.1 mol %). Thus, certain embodiments of the invention provide atherapeutic agent-lipid particle based on formulation K, which comprisesat least one therapeutic agent described herein (e.g., a nucleic acidmolecule).

Exemplary lipid formulation L includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.2%), cationic lipid (52.0%), phospholipid (9.7%), cholesterol(36.2%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(2.2%), the cationic lipid is1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA; Compound(15)) (52.0%), the phospholipid is DSPC (9.7%), and cholesterol ispresent at 36.2%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or 0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation L, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation M includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.7%), cationic lipid (58.4%), phospholipid (13.1%), cholesterol(25.7%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(2.7%), the cationic lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA) (58.4%), the phospholipid is DPPC (13.1%), and cholesterol ispresent at 25.7%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation M, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation N includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(3.0%), cationic lipid (53.3%), phospholipid (12.1%), cholesterol(31.5%), wherein the actual amounts of the lipids present may vary byby, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(3.0%), the cationic lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA) (53.3%), the phospholipid is DPPC (12.1%), and cholesterol ispresent at 31.5%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation N, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation O includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.5%), cationic lipid (56.2%), phospholipid (7.8%), cholesterol(34.7%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(1.5%), the cationic lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA) (56.2%), the phospholipid is DPPC (7.8%), and cholesterol ispresent at 34.7%, wherein the actual amounts of the lipids present mayvary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation O, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation P includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.1%), cationic lipid (48.6%), phospholipid (15.5%), cholesterol(33.8%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(2.1%), the cationic lipid is3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine(DLin-MP-DMA; Compound (8)) (48.6%), the phospholipid is DSPC (15.5%),and cholesterol is present at 33.8%, wherein the actual amounts of thelipids present may vary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %).Thus, certain embodiments of the invention provide a therapeuticagent-lipid particle based on formulation P, which comprises at leastone therapeutic agent described herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation Q includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.5%), cationic lipid (57.9%), phospholipid (9.2%), cholesterol(30.3%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(2.5%), the cationic lipid is(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate (Compound (13)) (57.9%), the phospholipid isDSPC (9.2%), and cholesterol is present at 30.3%, wherein the actualamounts of the lipids present may vary by, e.g., ±5% (or e.g., ±4 mol %,±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or0.1 mol %). Thus, certain embodiments of the invention provide atherapeutic agent-lipid particle based on formulation Q, which comprisesat least one therapeutic agent described herein (e.g., a nucleic acidmolecule).

Exemplary lipid formulation R includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.6%), cationic lipid (54.6%), phospholipid (10.9%), cholesterol(32.8%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(1.6%), the cationic lipid is3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine(Compound (8)) (54.6%), the phospholipid is DSPC (10.9%), andcholesterol is present at 32.8%, wherein the actual amounts of thelipids present may vary by, e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %).Thus, certain embodiments of the invention provide a therapeuticagent-lipid particle based on formulation R, which comprises at leastone therapeutic agent described herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation S includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.9%), cationic lipid (49.6%), phospholipid (16.3%), cholesterol(31.3%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(2.9%), the cationic lipid is(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate (Compound (13)) (49.6%), the phospholipid isDPPC (16.3%), and cholesterol is present at 31.3%, wherein the actualamounts of the lipids present may vary by, e.g., ±5% (or e.g., ±4 mol %,±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or±0.1 mol %). Thus, certain embodiments of the invention provide atherapeutic agent-lipid particle based on formulation S, which comprisesat least one therapeutic agent described herein (e.g., a nucleic acidmolecule).

Exemplary lipid formulation T includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(0.7%), cationic lipid (50.5%), phospholipid (8.9%), cholesterol(40.0%), wherein the actual amounts of the lipids present may vary by,e.g., ±5% (or e.g., ±4 mol %, ±3 mol %, +2 mol %, +1 mol %, +0.75 mol %,+0.5 mol %, +0.25 mol %, or +0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(0.7%), the cationic lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA) (50.5%), the phospholipid is DPPC (8.9%), and cholesterol ispresent at 40.0%, wherein the actual amounts of the lipids present mayvary by, e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %,+0.75 mol %, +0.5 mol %, +0.25 mol %, or +0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation T, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation U includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.0%), cationic lipid (51.4%), phospholipid (15.0%), cholesterol(32.6%), wherein the actual amounts of the lipids present may vary by,e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %, +0.75 mol %,+0.5 mol %, +0.25 mol %, or +0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(1.0%), the cationic lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA) (51.4%), the phospholipid is DSPC (15.0%), and cholesterol ispresent at 32.6%, wherein the actual amounts of the lipids present mayvary by, e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %,+0.75 mol %, +0.5 mol %, +0.25 mol %, or +0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation U, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation V includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.3%), cationic lipid (60.0%), phospholipid (7.2%), cholesterol(31.5%), wherein the actual amounts of the lipids present may vary by,e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %, +0.75 mol %,+0.5 mol %, +0.25 mol %, or +0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(1.3%), the cationic lipid is1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA) (60.0%), thephospholipid is DSPC (7.2%), and cholesterol is present at 31.5%,wherein the actual amounts of the lipids present may vary by, e.g., +5%(or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %, +0.75 mol %, +0.5 mol%, +0.25 mol %, or +0.1 mol %). Thus, certain embodiments of theinvention provide a therapeutic agent-lipid particle based onformulation V, which comprises at least one therapeutic agent describedherein (e.g., a nucleic acid molecule).

Exemplary lipid formulation W includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(1.8%), cationic lipid (51.6%), phospholipid (8.4%), cholesterol(38.3%), wherein the actual amounts of the lipids present may vary by,e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %, +0.75 mol %,+0.5 mol %, +0.25 mol %, or +0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(1.8%), the cationic lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA) (51.6%), the phospholipid is DSPC (8.4%), and cholesterol ispresent at 38.3%, wherein the actual amounts of the lipids present mayvary by, e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %,+0.75 mol %, +0.5 mol %, +0.25 mol %, or +0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation W, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation X includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.4%), cationic lipid (48.5%), phospholipid (10.0%), cholesterol(39.2%), wherein the actual amounts of the lipids present may vary by,e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %, +0.75 mol %,+0.5 mol %, +0.25 mol %, or +0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(2.4%), the cationic lipid is1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA; Compound(15)) (48.5%), the phospholipid is DPPC (10.0%), and cholesterol ispresent at 39.2%, wherein the actual amounts of the lipids present mayvary by, e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %,+0.75 mol %, +0.5 mol %, +0.25 mol %, or +0.1 mol %). Thus, certainembodiments of the invention provide a therapeutic agent-lipid particlebased on formulation X, which comprises at least one therapeutic agentdescribed herein (e.g., a nucleic acid molecule).

Exemplary lipid formulation Y includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.6%), cationic lipid (61.2%), phospholipid (7.1%), cholesterol(29.2%), wherein the actual amounts of the lipids present may vary by,e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %, +0.75 mol %,+0.5 mol %, +0.25 mol %, or +0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DMA (compound (66))(2.6%), the cationic lipid is(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate (Compound (13)) (61.2%), the phospholipid isDSPC (7.1%), and cholesterol is present at 29.2%, wherein the actualamounts of the lipids present may vary by, e.g., ±5% (or e.g., ±4 mol %,±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or±0.1 mol %). Thus, certain embodiments of the invention provide atherapeutic agent-lipid particle based on formulation Y, which comprisesat least one therapeutic agent described herein (e.g., a nucleic acidmolecule).

Exemplary lipid formulation Z includes the following components (whereinthe percentage values of the components are mole percent): PEG-lipid(2.2%), cationic lipid (49.7%), phospholipid (12.1%), cholesterol(36.0%), wherein the actual amounts of the lipids present may vary by,e.g., +5% (or e.g., +4 mol %, +3 mol %, +2 mol %, +1 mol %, +0.75 mol %,+0.5 mol %, +0.25 mol %, or +0.1 mol %). For example, in onerepresentative embodiment, the PEG-lipid is PEG-C-DOMG (compound (67))(2.2%), the cationic lipid is(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) (Compound (7)) (49.7%), the phospholipid isDPPC (12.1%), and cholesterol is present at 36.0%, wherein the actualamounts of the lipids present may vary by, e.g., ±5% (or e.g., ±4 mol %,±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or0.1 mol %). Thus, certain embodiments of the invention provide atherapeutic agent-lipid particle based on formulation Z, which comprisesat least one therapeutic agent described herein (e.g., a nucleic acidmolecule).

Accordingly, certain embodiments of the invention provide a therapeuticagent-lipid particle described herein, wherein the lipids are formulatedas described in any one of formulations A, B, C, D, E, F, G, H, I, J, K,L, M, N, O, P, Q, R, S, T, U, V, W, X, Y or Z.

Preparation of Lipid Particles

The therapeutic agent-lipid particles, in which a therapeutic agent(e.g., a nucleic acid, such as a siRNA or mRNA) is entrapped within thelipid portion of the particle and is protected from degradation, can beformed by any method known in the art including, but not limited to, acontinuous mixing method, a direct dilution process, and an in-linedilution process.

In particular embodiments, the cationic lipids may comprise lipids ofFormula I-III or salts thereof, alone or in combination with othercationic lipids. In other embodiments, the non-cationic lipids are eggsphingomyelin (ESM), distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC),1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphati dylethanolamine,14:0 PE (1,2-dimyri stoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE (1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphati dylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol,derivatives thereof, or combinations thereof.

In certain embodiments, the therapeutic agent-lipid particles producedvia a continuous mixing method, e.g., a process that includes providingan aqueous solution comprising a therapeutic agent in a first reservoir,providing an organic lipid solution in a second reservoir (wherein thelipids present in the organic lipid solution are solubilized in anorganic solvent, e.g., a lower alkanol such as ethanol), and mixing theaqueous solution with the organic lipid solution such that the organiclipid solution mixes with the aqueous solution so as to substantiallyinstantaneously produce a lipid vesicle (e.g., liposome) encapsulatingthe therapeutic agent within the lipid vesicle. This process and theapparatus for carrying out this process are described in detail in U.S.Patent Publication No. 20040142025, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a lipid vesicle substantially instantaneously upon mixing. Asused herein, the phrase “continuously diluting a lipid solution with abuffer solution” (and variations) generally means that the lipidsolution is diluted sufficiently rapidly in a hydration process withsufficient force to effectuate vesicle generation. By mixing the aqueoussolution comprising a therapeutic agent with the organic lipid solution,the organic lipid solution undergoes a continuous stepwise dilution inthe presence of the buffer solution (i.e., aqueous solution) to producea therapeutic agent-lipid particle.

The therapeutic agent-lipid particles formed using the continuous mixingmethod typically have a size of from about 30 nm to about 150 nm, fromabout 40 nm to about 150 nm, from about 50 nm to about 150 nm, fromabout 60 nm to about 130 nm, from about 70 nm to about 110 nm, fromabout 70 nm to about 100 nm, from about 80 nm to about 100 nm, fromabout 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, or 150 nm (or any fraction thereof or range therein). Theparticles thus formed do not aggregate and are optionally sized toachieve a uniform particle size.

In another embodiment, the therapeutic agent-lipid particles producedvia a direct dilution process that includes forming a lipid vesicle(e.g., liposome) solution and immediately and directly introducing thelipid vesicle solution into a collection vessel containing a controlledamount of dilution buffer. In preferred aspects, the collection vesselincludes one or more elements configured to stir the contents of thecollection vessel to facilitate dilution. In one aspect, the amount ofdilution buffer present in the collection vessel is substantially equalto the volume of lipid vesicle solution introduced thereto. As anon-limiting example, a lipid vesicle solution in 45% ethanol whenintroduced into the collection vessel containing an equal volume ofdilution buffer will advantageously yield smaller particles.

In yet another embodiment, the therapeutic agent-lipid particlesproduced via an in-line dilution process in which a third reservoircontaining dilution buffer is fluidly coupled to a second mixing region.In this embodiment, the lipid vesicle (e.g., liposome) solution formedin a first mixing region is immediately and directly mixed with dilutionbuffer in the second mixing region. In preferred aspects, the secondmixing region includes a T-connector arranged so that the lipid vesiclesolution and the dilution buffer flows meet as opposing 180° flows;however, connectors providing shallower angles can be used, e.g., fromabout 27° to about 180° (e.g., about 90°). A pump mechanism delivers acontrollable flow of buffer to the second mixing region. In one aspect,the flow rate of dilution buffer provided to the second mixing region iscontrolled to be substantially equal to the flow rate of lipid vesiclesolution introduced thereto from the first mixing region. Thisembodiment advantageously allows for more control of the flow ofdilution buffer mixing with the lipid vesicle solution in the secondmixing region, and therefore also the concentration of lipid vesiclesolution in buffer throughout the second mixing process. Such control ofthe dilution buffer flow rate advantageously allows for small particlesize formation at reduced concentrations.

These processes and the apparatuses for carrying out these directdilution and in-line dilution processes are described in detail in U.S.Patent Publication No. 20070042031, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The therapeutic agent-lipid particles formed using the direct dilutionand in-line dilution processes typically have a size of from about 30 nmto about 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 nm to about 100 nm, from about 80 nm toabout 100 nm, from about 90 nm to about 100 nm, from about 70 to about90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm,35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, or 150 nm (or any fraction thereof or rangetherein). The particles thus formed do not aggregate and are optionallysized to achieve a uniform particle size.

The lipid particles can be sized by any of the methods available forsizing liposomes. The sizing may be conducted in order to achieve adesired size range and relatively narrow distribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the therapeutic agents present in the particles(e.g., nucleic acid molecules) are precondensed as described in, e.g.,U.S. patent application Ser. No. 09/744,103, the disclosure of which isherein incorporated by reference in its entirety for all purposes.

In other embodiments, the methods may further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brand name POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In some embodiments, the therapeutic agent (e.g., nucleic acid, such assiRNA or mRNA) to lipid ratios (mass/mass ratios) in a formedtherapeutic agent-lipid particle will range from about 0.01 to about0.2, from about 0.05 to about 0.2, from about 0.02 to about 0.1, fromabout 0.03 to about 0.1, or from about 0.01 to about 0.08. The ratio ofthe starting materials (input) also falls within this range. In otherembodiments, the particle preparation uses about 400 μg therapeuticagent per 10 mg total lipid or a therapeutic agent to lipid mass ratioof about 0.01 to about 0.08 and, more preferably, about 0.04, whichcorresponds to 1.25 mg of total lipid per 50 μg of therapeutic agent. Inother preferred embodiments, the particle has a therapeutic agent:lipidmass ratio of about 0.08.

In other embodiments, the lipid to therapeutic agent (e.g., nucleicacid, such as siRNA or mRNA) ratios (mass/mass ratios) in a formedtherapeutic agent-lipid particle will range from about 1 (1:1) to about100 (100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1)to about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3(3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), fromabout 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1),from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 25(25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) toabout 25 (25:1), from about 5 (5:1) to about 20 (20:1), from about 5(5:1) to about 15 (15:1), from about 5 (5:1) to about 10 (10:1), orabout 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9 (9:1), 10 (10:1), 11 (11:1),12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1), 16 (16:1), 17 (17:1), 18(18:1), 19 (19:1), 20 (20:1), 21 (21:1), 22 (22:1), 23 (23:1), 24(24:1), or 25 (25:1), or any fraction thereof or range therein. Theratio of the starting materials (input) also falls within this range.

As previously discussed, the conjugated lipid may further include a CPL.A variety of general methods for making lipid particle-CPLs(CPL-containing lipid particles) are discussed herein. Two generaltechniques include the “post-insertion” technique, that is, insertion ofa CPL into, for example, a pre-formed lipid particle, and the “standard”technique, wherein the CPL is included in the lipid mixture during, forexample, the lipid particle formation steps. The post-insertiontechnique results in lipid particles having CPLs mainly in the externalface of the lipid particle bilayer membrane, whereas standard techniquesprovide lipid particles having CPLs on both internal and external faces.The method is especially useful for vesicles made from phospholipids(which can contain cholesterol) and also for vesicles containingPEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of making lipidparticle-CPLs are taught, for example, in U.S. Pat. Nos. 5,705,385;6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent PublicationNo. 20020072121; and PCT Publication No. WO 00/62813, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

Additional Carrier Systems

Non-limiting examples of additional lipid-based carrier systems suitablefor use include lipoplexes (see, e.g., U.S. Patent Publication No.20030203865; and Zhang et al., J. Control Release, 100:165-180 (2004)),pH-sensitive lipoplexes (see, e.g., U.S. Patent Publication No.20020192275), reversibly masked lipoplexes (see, e.g., U.S. PatentPublication Nos. 20030180950), cationic lipid-based compositions (see,e.g., U.S. Pat. No. 6,756,054; and U.S. Patent Publication No.20050234232), cationic liposomes (see, e.g., U.S. Patent PublicationNos. 20030229040, 20020160038, and 20020012998; U.S. Pat. No. 5,908,635;and PCT Publication No. WO 01/72283), anionic liposomes (see, e.g., U.S.Patent Publication No. 20030026831), pH-sensitive liposomes (see, e.g.,U.S. Patent Publication No. 20020192274; and AU 2003210303),antibody-coated liposomes (see, e.g., U.S. Patent Publication No.20030108597; and PCT Publication No. WO 00/50008), cell-type specificliposomes (see, e.g., U.S. Patent Publication No. 20030198664),liposomes containing nucleic acid and peptides (see, e.g., U.S. Pat. No.6,207,456), liposomes containing lipids derivatized with releasablehydrophilic polymers (see, e.g., U.S. Patent Publication No.20030031704), lipid-entrapped nucleic acid (see, e.g., PCT PublicationNos. WO 03/057190 and WO 03/059322), lipid-encapsulated nucleic acid(see, e.g., U.S. Patent Publication No. 20030129221; and U.S. Pat. No.5,756,122), other liposomal compositions (see, e.g., U.S. PatentPublication Nos. 20030035829 and 20030072794; and U.S. Pat. No.6,200,599), stabilized mixtures of liposomes and emulsions (see, e.g.,EP1304160), emulsion compositions (see, e.g., U.S. Pat. No. 6,747,014),and nucleic acid micro-emulsions (see, e.g., U.S. Patent Publication No.20050037086).

Examples of polymer-based carrier systems suitable for use include, butare not limited to, cationic polymer-nucleic acid complexes (i.e.,polyplexes). To form a polyplex, a nucleic acid (e.g., a siRNA moleculeor mRNA molecule) is typically complexed with a cationic polymer havinga linear, branched, star, or dendritic polymeric structure thatcondenses the nucleic acid into positively charged particles capable ofinteracting with anionic proteoglycans at the cell surface and enteringcells by endocytosis. In some embodiments, the polyplex comprisesnucleic acid complexed with a cationic polymer such as polyethylenimine(PEI) (see, e.g., U.S. Pat. No. 6,013,240; commercially available fromQbiogene, Inc. (Carlsbad, Calif.) as In vivo jetPEF™, a linear form ofPEI), polypropylenimine (PPI), polyvinylpyrrolidone (PVP), poly-L-lysine(PLL), diethylaminoethyl (DEAE)-dextran, poly(β-amino ester) (PAE)polymers (see, e.g., Lynn et al., J. Am. Chem. Soc., 123:8155-8156(2001)), chitosan, polyamidoamine (PAMAM) dendrimers (see, e.g.,Kukowska-Latallo et al., Proc. Natl. Acad. Sci. USA, 93:4897-4902(1996)), porphyrin (see, e.g., U.S. Pat. No. 6,620,805), polyvinylether(see, e.g., U.S. Patent Publication No. 20040156909), polycyclicamidinium (see, e.g., U.S. Patent Publication No. 20030220289), otherpolymers comprising primary amine, imine, guanidine, and/or imidazolegroups (see, e.g., U.S. Pat. No. 6,013,240; PCT Publication No.WO/9602655; PCT Publication No. WO95/21931; Zhang et al., J. ControlRelease, 100:165-180 (2004); and Tiera et al., Curr. Gene Ther., 6:59-71(2006)), and a mixture thereof. In other embodiments, the polyplexcomprises cationic polymer-nucleic acid complexes as described in U.S.Patent Publication Nos. 20060211643, 20050222064, 20030125281, and20030185890, and PCT Publication No. WO 03/066069; biodegradablepoly(β-amino ester) polymer-nucleic acid complexes as described in U.S.Patent Publication No. 20040071654; microparticles containing polymericmatrices as described in U.S. Patent Publication No. 20040142475; othermicroparticle compositions as described in U.S. Patent Publication No.20030157030; condensed nucleic acid complexes as described in U.S.Patent Publication No. 20050123600; and nanocapsule and microcapsulecompositions as described in AU 2002358514 and PCT Publication No. WO02/096551.

In certain instances, a nucleic acid may be complexed with cyclodextrinor a polymer thereof. Non-limiting examples of cyclodextrin-basedcarrier systems include the cyclodextrin-modified polymer-nucleic acidcomplexes described in U.S. Patent Publication No. 20040087024; thelinear cyclodextrin copolymer-nucleic acid complexes described in U.S.Pat. Nos. 6,509,323, 6,884,789, and 7,091,192; and the cyclodextrinpolymer-complexing agent-nucleic acid complexes described in U.S. Pat.No. 7,018,609. In certain other instances, a nucleic acid may becomplexed with a peptide or polypeptide. An example of a protein-basedcarrier system includes, but is not limited to, the cationicoligopeptide-nucleic acid complex described in PCT Publication No.WO95/21931.

Administration of Lipid Particles

The lipid particles (e.g., a therapeutic agent-lipid particle, such as anucleic-acid lipid particle) can be adsorbed to almost any cell typewith which they are mixed or contacted. Once adsorbed, the particles caneither be endocytosed by a portion of the cells, exchange lipids withcell membranes, or fuse with the cells. Transfer or incorporation of thetherapeutic agent portion of the particle can take place via any one ofthese pathways. In particular, when fusion takes place, the particlemembrane is integrated into the cell membrane and the contents of theparticle combine with the intracellular fluid.

The lipid particles (e.g., therapeutic agent-lipid particles) can beadministered either alone or in a mixture with a pharmaceuticallyacceptable carrier (e.g., physiological saline or phosphate buffer)selected in accordance with the route of administration and standardpharmaceutical practice. Generally, normal buffered saline (e.g.,135-150 mM NaCl) will be employed as the pharmaceutically acceptablecarrier. Other suitable carriers include, e.g., water, buffered water,0.4% saline, 0.3% glycine, and the like, including glycoproteins forenhanced stability, such as albumin, lipoprotein, globulin, etc.Additional suitable carriers are described in, e.g., REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). As used herein, “carrier” includes any and allsolvents, dispersion media, vehicles, coatings, diluents, antibacterialand antifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The phrase“pharmaceutically acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human.

The pharmaceutically acceptable carrier is generally added followinglipid particle formation. Thus, after the lipid particle is formed, theparticle can be diluted into pharmaceutically acceptable carriers suchas normal buffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions may be sterilized by conventional,well-known sterilization techniques. Aqueous solutions can be packagedfor use or filtered under aseptic conditions and lyophilized, thelyophilized preparation being combined with a sterile aqueous solutionprior to administration. The compositions can contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, and calcium chloride.Additionally, the particle suspension may include lipid-protectiveagents which protect lipids against free-radical and lipid-peroxidativedamages on storage. Lipophilic free-radical quenchers, such asalphatocopherol, and water-soluble iron-specific chelators, such asferrioxamine, are suitable.

In Vivo Administration

Systemic delivery for in vivo therapy, e.g., delivery of a therapeuticagent described herein, such as a nucleic acid, to a distal target cellvia body systems such as the circulation, has been achieved usingtherapeutic agent-lipid particles such as those described in PCTPublication Nos. WO 05/007196, WO 05/121348, WO 05/120152, and WO04/002453, the disclosures of which are herein incorporated by referencein their entirety for all purposes.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71(1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

In embodiments where the lipid particles are administered intravenously,at least about 5%, 10%, 15%, 20%, or 25% of the total injected dose ofthe particles is present in plasma about 8, 12, 24, 36, or 48 hoursafter injection. In other embodiments, more than about 20%, 30%, 40% andas much as about 60%, 70% or 80% of the total injected dose of the lipidparticles is present in plasma about 8, 12, 24, 36, or 48 hours afterinjection. In certain instances, more than about 10% of a plurality ofthe particles is present in the plasma of a mammal about 1 hour afteradministration. In certain other instances, the presence of the lipidparticles is detectable at least about 1 hour after administration ofthe particle. In some embodiments, the presence of a therapeutic agent,such as a nucleic acid molecule, is detectable in cells at about 8, 12,24, 36, 48, 60, 72 or 96 hours after administration. In someembodiments, the effect of a therapeutic agent, such as a nucleic acidmolecule, is detectable in cells at about 8, 12, 24, 36, 48, 60, 72 or96 hours after administration. In other embodiments, downregulation ofexpression of a target sequence, such as a viral or host sequence, by asiRNA molecule is detectable at about 8, 12, 24, 36, 48, 60, 72 or 96hours after administration. In yet other embodiments, downregulation ofexpression of a target sequence, such as a viral or host sequence, by asiRNA molecule occurs preferentially in infected cells and/or cellscapable of being infected. In further embodiments, the presence oreffect of a therapeutic agent in cells at a site proximal or distal tothe site of administration is detectable at about 12, 24, 48, 72, or 96hours, or at about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28days after administration. In additional embodiments, the lipidparticles are administered parenterally or intraperitoneally.

The compositions, either alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation (e.g., intranasally orintratracheally) (see, Brigham et al., Am. J. Sci., 298:278 (1989)).Aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier.Suitable formulations are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic agent(e.g., a nucleic acid molecule) suspended in diluents such as water,saline, or PEG 400; (b) capsules, sachets, or tablets, each containing apredetermined amount of a therapeutic agent, as liquids, solids,granules, or gelatin; (c) suspensions in an appropriate liquid; and (d)suitable emulsions. Tablet forms can include one or more of lactose,sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potatostarch, microcrystalline cellulose, gelatin, colloidal silicon dioxide,talc, magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers. Lozenge forms can comprise atherapeutic agent in a flavor, e.g., sucrose, as well as pastillescomprising the therapeutic agent in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the therapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining therapeutic agent-lipid particles can be formulated andadministered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

The amount of particles administered will depend upon the ratio oftherapeutic agent to lipid, the particular therapeutic agent used, thedisease or condition being treated, the age, weight, and condition ofthe patient, and the judgment of the clinician, but will generally bebetween about 0.01 and about 50 mg per kilogram of body weight,preferably between about 0.1 and about 5 mg/kg of body weight, or about10⁸-10¹⁰ particles per administration (e.g., injection).

In certain embodiments, the therapeutic agent is administered via atherapeutic agent lipid particle.

In certain embodiments, with respect to methods that include the use ofa cocktail of therapeutic agents (e.g., a cocktail of nucleic acidmolecules) encapsulated within lipid particles, the differenttherapeutic agents are co-encapsulated in the same lipid particle.

In certain embodiments, the with respect to methods that include the useof a cocktail of therapeutic agents (e.g., a cocktail of nucleic acidmolecules) encapsulated within lipid particles, each type of therapeuticagent species present in the cocktail is encapsulated in its ownparticle.

In certain embodiments, the with respect to methods that include the useof a cocktail of therapeutic agents (e.g., a cocktail of nucleic acidmolecules) encapsulated within lipid particles, some therapeutic agentspecies are co-encapsulated in the same particle while other therapeuticagent species are encapsulated in different particles.

Formulation and Administration of NSAID and/or Additional TherapeuticAgent(s) As discussed herein, certain embodiments of the inventionprovide for the administration of a NSAID prior to at least one lipidformulated therapeutic agent being administered. In certain embodimentsof the invention, a NSAID is administered via injection and at least onelipid formulated therapeutic agent is intravenously administered (i.e.,administered sequentially in order). Additionally, in certainembodiments, one or more additional therapeutic agents may also beadministered (e.g., simultaneously or sequentially with the NSAID and/orthe at least one lipid formulated therapeutic agent). Thus, these agentsmay be administered to a mammal as indicated below.

It will be understood that agents can be formulated together in a singlepreparation or that they can be formulated separately and, thus,administered separately, either simultaneously or sequentially. In oneembodiment, when the agents are administered sequentially (e.g. atdifferent times), the agents may be administered so that theirbiological effects overlap (i.e. each agent is producing a biologicaleffect at a single given time).

The agents can be formulated for and administered using any acceptableroute of administration depending on the agent selected. For example,suitable routes include, but are not limited to, oral, sublingual,buccal, topical, transdermal, parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. In one embodiment, the small moleculeagents identified herein can be administered orally or by injection(e.g., into a blood vessel, such as a vein). In another embodiment, thenucleic acid molecules can be administered by injection (e.g., into ablood vessel, such as a vein), or subcutaneously.

Typically, the NSAID is administered via injection and the at least onelipid formulated therapeutic agent is administered intravenously. Incertain embodiments, the NSAID is administered parenterally. In certainembodiments, the NSAID is administered intravenously.

In certain embodiments, the NSAID is administered intramuscularly. Incertain embodiments, the NSAID is administered subcutaneously.

The agents can be individually formulated by mixing at ambienttemperature at the appropriate pH, and at the desired degree of purity,with physiologically acceptable carriers, i.e., carriers that arenon-toxic to recipients at the dosages and concentrations employed. ThepH of the formulation depends mainly on the particular use and theconcentration of compound, but may typically range anywhere from about 3to about 8. The agents ordinarily will be stored as a solid composition,although lyophilized formulations or aqueous solutions are acceptable.

Compositions comprising the agents can be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disease orcondition being treated, the particular mammal being treated, theclinical condition of the individual patient, the cause of the disorder,the site of administration, the method of administration, the schedulingof administration, and other factors known to medical practitioners.

The agents may be administered in any convenient administrative form,e.g., tablets, powders, capsules, solutions, dispersions, suspensions,syrups, sprays, suppositories, gels, emulsions, patches, etc. Suchcompositions may contain components conventional in pharmaceuticalpreparations, e.g., diluents, carriers, pH modifiers, sweeteners,bulking agents, and further active agents. If parenteral administrationis desired, the compositions will be sterile and in a solution orsuspension form suitable for injection or infusion.

Suitable carriers and excipients are well known to those skilled in theart and are described in detail in, e.g., Ansel, Howard C., et al.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems.Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R.,et al. Remington: The Science and Practice of Pharmacy. Philadelphia:Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook ofPharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. Theformulations may also include one or more buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents, diluents and other known additives to provide an elegantpresentation of the drug or aid in the manufacturing of thepharmaceutical product (i.e., medicament).

The agents are typically dosed at least at a level to reach the desiredbiological effect. Thus, an effective dosing regimen will dose at leasta minimum amount that reaches the desired biological effect, orbiologically effective dose, however, the dose should not be so high asto outweigh the benefit of the biological effect with unacceptable sideeffects. Therefore, an effective dosing regimen will dose no more thanthe maximum tolerated dose (“MTD”). The maximum tolerated dose isdefined as the highest dose that produces an acceptable incidence ofdose-limiting toxicities (“DLT”). Doses that cause an unacceptable rateof DLT are considered non-tolerated. Typically, the MTD for a particularschedule is established in phase 1 clinical trials. These are usuallyconducted in patients by starting at a safe starting dose of 1/10 thesevere toxic dose (“STD10”) in rodents (on a mg/m² basis) and accruingpatients in cohorts of three, escalating the dose according to amodified Fibonacci sequence in which ever higher escalation steps haveever decreasing relative increments (e.g., dose increases of 100%, 65%,50%, 40%, and 30% to 35% thereafter). The dose escalation is continuedin cohorts of three patients until a non-tolerated dose is reached. Thenext lower dose level that produces an acceptable rate of DLT isconsidered to be the MTD.

The amount of the agents administered will depend upon the particularagent used, the disease or condition being treated, the age, weight, andcondition of the patient, and the judgment of the clinician, but willgenerally be between about 0.2 to 2.0 grams per day. For example, incertain embodiments, the NSAID is administered in an approved dosage forinjection. In certain embodiments, the NSAID is administered in a doseof about 1 mg to about 1000 mg. In certain embodiments, a dose of theNSAID is administered one or more times per day. For example, in certainembodiments, the NSAID is ketorolac formulated for injection, wherein asingle dose ranging between about 15 mg to about 60 mg is administered(e.g., about 30 mg to about 60 mg) or wherein a dose of about 15 mg toabout 30 mg is administered multiple times in a day (e.g., a dose isadministered every 6 hrs, not exceeding about 60 mg to about 120mg/day).

Administration Regimen

NSAID in Combination with at Least One Lipid Formulated TherapeuticAgent

It will be understood that the NSAID and the at least one lipidformulated therapeutic agent are formulated separately and administeredsequentially (the NSAID is administered prior to the administration ofthe at least one lipid formulated therapeutic agent). For purposes ofthe present disclosure, such administration regimens are considered theadministration of an NSAID “in combination with” at least one lipidformulated therapeutic agent.

As described herein, the NSAID is administered prior to at least onelipid formulated therapeutic agent being administered. For example, afirst component may be deemed to be administered “prior to” a secondcomponent if the first component is administered 1 week before, 72 hoursbefore, 60 hours before, 48 hours before, 36 hours before, 24 hoursbefore, 12 hours before, 6 hours before, 5 hours before, 4 hours before,3 hours before, 2 hours before, 1 hour before, 59, 58, 57, 56, 55, 54,53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36,35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 minutes before,or 1 or less than 1 minute before administration of the secondcomponent. In certain embodiments, the NSAID is administered withinabout 3 hours prior to the at least one lipid formulated therapeuticagent being administered. In certain embodiments, the NSAID isadministered within about 2 hours prior to the at least one lipidformulated therapeutic agent being administered. In certain embodiments,the NSAID is administered within about 1 hour prior to the at least onelipid formulated therapeutic agent being administered. In certainembodiments, the NSAID is administered within about 30 minutes prior tothe at least one lipid formulated therapeutic agent being administered.In certain embodiments, the NSAID is administered within about 20minutes prior to the at least one lipid formulated therapeutic agentbeing administered. In certain embodiments, the NSAID is administeredwithin about 10 minutes prior to the at least one lipid formulatedtherapeutic agent being administered. For example, in certainembodiments, the NSAID is administered intramuscularly or subcutaneouslywithin about 1 hour prior to the at least one lipid formulatedtherapeutic agent being administered. In certain embodiments, the NSAIDis administered intravenously within about 1-2 hours prior to the atleast one lipid formulated therapeutic agent being administered.

In certain embodiments, the NSAID is administered at least once prior tothe administration of the at least one lipid formulated therapeuticagent. In certain embodiments, the NSAID is administered once prior tothe administration of the at least one lipid formulated therapeuticagent.

Administration Regimen of an Additional Therapeutic Agent in Combinationwith a NSAID or a Lipid Formulated Therapeutic Agent

It will be understood that a NSAID and at least one additionaltherapeutic agent can be formulated together in a single preparation orthat they can be formulated separately and, thus, administeredseparately, either simultaneously or sequentially (the agent(s) may beadministered prior to or after the administration of the NSAID). In oneembodiment, when the agents are administered sequentially (e.g. atdifferent times), the agents may be administered so that theirbiological effects overlap (i.e. each agent is producing a biologicaleffect at a single given time). For purposes of the present disclosure,such administration regimens are considered the administration of aNSAID “in combination with” at least one additional therapeutic agent oractive component.

It will also be understood that at least one lipid formulatedtherapeutic agent and at least one additional therapeutic agent can beformulated together in a single preparation or that they can beformulated separately and, thus, administered separately, eithersimultaneously or sequentially (the agent(s) may be administered priorto or after the administration of the at least one lipid formulatedtherapeutic agent). In one embodiment, when the agents are administeredsequentially (e.g. at different times), the agents may be administeredso that their biological effects overlap (i.e. each agent is producing abiological effect at a single given time). For purposes of the presentdisclosure, such administration regimens are considered theadministration of at least one lipid formulated therapeutic agent “incombination with” at least one additional therapeutic agent or activecomponent.

The at least one additional therapeutic agent (additional component) maybe administered to a subject prior to administration of a NSAID or atleast one lipid formulated therapeutic agent. For example, a firstcomponent may be deemed to be administered “prior to” a second componentif the first component is administered 1 week before, 72 hours before,60 hours before, 48 hours before, 36 hours before, 24 hours before, 12hours before, 6 hours before, 5 hours before, 4 hours before, 3 hoursbefore, 2 hours before, 1 hour before, 30 minutes before, 15 minutesbefore, 10 minutes before, 5 minutes before, or less than 1 minutebefore administration of the second component. In other embodiments, theat least one additional therapeutic agent/component may be administeredto a subject after administration of a NSAID or at least one lipidformulated therapeutic agent. For example, a first component may bedeemed to be administered “after” a second component if the firstcomponent is administered 1 minute after, 5 minutes after, 10 minutesafter, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after,3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hoursafter, 24 hours after, 36 hours after, 48 hours after, 60 hours after,72 hours after administration of the second component. In certainembodiments, the additional therapeutic agent is a NSAID, which isadministered after the administration of the at least one lipidformulated therapeutic agent (e.g., between about 4 to about 8 hoursafter the lipid formulated therapeutic agent is administered). Incertain embodiments, the additional NSAID may be the same or differentfrom the NSAID administered via injection and may be administered by thesame or different route.

In yet other embodiments, the at least one additional therapeutic agentmay be administered to a subject concurrent with administration of aNSAID or at least one lipid formulated therapeutic agent. “Concurrent”administration, for purposes of the present invention, includes, e.g.,administration of a NSAID or at least one at least one lipid formulatedtherapeutic agent and the at least one additional therapeutic agent to asubject in a single dosage form (e.g., co-formulated), or in separatedosage forms administered to the subject within about 30 minutes or lessof each other. If administered in separate dosage forms, each dosageform may be administered via the same route (e.g., both the NSAID/lipidformulated therapeutic agent and the additional therapeutically activecomponent may be administered intravenously); alternatively, each dosageform may be administered via a different route (e.g., the NSAID may beadministered intramuscularly, the lipid formulated therapeutic agent maybe administered intravenously, and the additional therapeutically activecomponent may be administered orally). In any event, administering thecomponents in a single dosage from, in separate dosage forms by the sameroute, or in separate dosage forms by different routes are allconsidered “concurrent administration” for purposes of the presentdisclosure. For purposes of the present disclosure, administration of aNSAID/lipid formulated therapeutic agent “prior to”, “concurrent with,”or “after” (as those terms are defined herein above) administration ofan additional therapeutic agent is considered administration of anNSAID/lipid formulated therapeutic agent “in combination with” anadditional therapeutic agent).

As described herein, pharmaceutical compositions in which a NSAID orlipid formulated therapeutic agent is co-formulated with at least oneadditional therapeutic agent using a variety of dosage combinations mayalso be used.

Kits

One embodiment provides a kit. The kit may comprise a containercomprising the combination. Suitable containers include, for example,bottles, vials, syringes, blister pack, etc. The container may be formedfrom a variety of materials such as glass or plastic. The container mayhold the combination which is effective for treating the condition andmay have a sterile access port (for example, the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle).

The kit may further comprise a label or package-insert on or associatedwith the container. The term “package-insert” is used to refer toinstructions customarily included in commercial packages of therapeuticagents that contain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch therapeutic agents. In one embodiment, the label or package insertsindicates the administration of the NSAID via injection prior to theintravenous administration of the at least one lipid formulatedtherapeutic agent, for ameliorating an infusion reaction associated withthe intravenous administration of the at least one lipid formulatedtherapeutic agent.

In certain embodiments, the kits are suitable for the delivery of solidoral forms of the therapeutic agents, such as tablets or capsules. Sucha kit preferably includes a number of unit dosages. Such kits caninclude a card having the dosages oriented in the order of theirintended use. An example of such a kit is a “blister pack”. Blisterpacks are well known in the packaging industry and are widely used forpackaging pharmaceutical unit dosage forms. If desired, a memory aid canbe provided, for example in the form of numbers, letters, or othermarkings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to another embodiment, a kit may comprise (a) a firstcontainer with one agent contained therein (e.g., a NSAID); and (b) asecond container with a second agent contained therein (e.g., a lipidformulated therapeutic agent). Alternatively, or additionally, the kitmay further comprise a third container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The kit may further comprise directions for the administration of thetherapeutic agents. For example, the kit may further comprise directionsfor the simultaneous, sequential or separate administration of thetherapeutic agents to a patient in need thereof.

In certain other embodiments, the kit may comprise a container forcontaining separate compositions such as a divided bottle or a dividedfoil packet, however, the separate compositions may also be containedwithin a single, undivided container. In certain embodiments, the kitcomprises directions for the administration of the separate therapeuticagents. The kit form is particularly advantageous when the separatetherapeutic agents are preferably administered in different dosage forms(e.g., oral and parenteral), are administered at different dosageintervals, or when titration of the individual therapeutic agents of thecombination is desired by the prescribing physician.

In certain embodiments, the kit comprises a nonsteroidalanti-inflammatory (NSAID) and at least one lipid formulated therapeuticagent, a container, and a package insert or label indicating theadministration of the NSAID via injection prior to the intravenousadministration of the at least one lipid formulated therapeutic agent,for ameliorating an infusion reaction associated with the intravenousadministration of the at least one lipid formulated therapeutic agent.In certain embodiments, the kit comprises at least one additionaltherapeutic agent.

The invention will now be illustrated by the following non-limitingExamples.

Example 1

Evaluation of LNP in the Conscious Minipig

The objective of this study was to evaluate the effect of intravenousinfusion of LNP2 on hemodynamic function in the conscious, freely movingminipig, when administered with or without a bolus intravenous injectionof ketorolac or dexamethasone. The LNP formulation used in these studieshave the following lipid composition (molar ratios): PEG-lipid(PEG2000-C-DMA (1.1 mol %); Cationic lipid((6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethyl-amino)pentanoate (54.6 mol %); cholesterol (32.8 mol %); andDSPC (10.9 mol %). A lipid stock was prepared with the appropriatelipids dissolved in 90% ethanol (total concentration 12.24 mg/mL). ThesiRNA stock was made up in a 20 mM EDTA buffer at 1.215 mg/mL. The twostocks were combined using the Jeffs et al. method, blending in at-piece and the combined suspension diluted a further 3-fold withPhosphate Buffered Saline, pH 7.4. The sample was then concentrated andbuffer exchange performed via tangential flow ultrafiltration (MWCO 100k, GE Healthcare). The LNP were then sterile filtered (0.2 μm filter).Concentration was determined using RiboGreen Assay and a Varian CaryEclipse Fluorimeter. Particle size and polydispersity were determinedusing a Malvern Nano Series Zetasizer.

Methods

Four (4) treatment naïve female Gottingen minipigs (Marshall Farms,N.Y.) were surgically implanted with a D70-PCT PhysioTel implantableradiotelemetry transmitter (Data Sciences International, St. Paul,Minn.) to allow for the measurement of physical activity, bodytemperature, ECG, heart rate, and arterial blood pressure. The pigs wereallowed to recover from surgery for at least 7 days prior to use in thestudy.

Following surgical recovery, the same four pigs (15-20 kg) received a60-minute intravenous infusion of vehicle (0.9% saline), LNP2,LNP2+ketorolac, or LNP2+dexamethasone as outlined in the Table 1. Eachtreatment cohort was separated by a 7-day washout period.

TABLE 1 Lipid Dose siRNA Dose Dose Volume Cohort Treatment (mg/kg)(mg/kg) (mL/kg) 1 Vehicle 0 0 1 2 LNP2 0.3 3.5 1 3 LNP2 + ketorolac^(†)0.3 3.5 1 4 LNP2 + dexamethasone^(‡) 0.3 3.5 1 ^(†)Ketorolac wasadministered as an intravenous bolus dose (1 mg/kg), 10 minutes prior toinfusion of LNP2. ^(‡)Dexamethasone was administered as an intravenousbolus dose (0.3 mg/kg), 10 minutes prior to infusion of LNP2.

Just prior to each infusion and at approximately 1, 3, 6 and 24 hoursafter the start of each infusion, a mixed venous blood sample was taken,processed to plasma, stored frozen, and used for subsequent analysis ofthromboxane B2 (11-dehydrothromboxane B2). Heart rate and arterial bloodpressure was continuously recorded from 30 minutes prior to eachinfusion to 24 hours after the start of each infusion.

Results and Discussion

Administration of LNP2 resulted in a profound increase in plasmathromboxane B2 levels at 1 and 3 hours after the start of infusion. Theincrease in thromboxane B2 was absent in the vehicle control group, wasmitigated completely by co-administration with ketorolac, and wasreduced, but not completely, by co-administration with dexamethasone(FIG. 1).

The increase in thromboxane B2 appears to be associated with an overallincrease in blood pressure observed with LNP2 during the infusion andshortly thereafter (FIG. 2). The effect of LNP2 on blood pressure wasbiphasic with an almost immediate increase (˜15-20 mmHg) that occurredwithin minutes of starting the infusion, returning to baseline by 10minutes, and then gradually increasing, reaching a peak increase ofapproximately 40 mmHg by 80-85 minutes after the start of infusion. Itis noteworthy that this acute infusion reaction to LNP2 was completelyprevented by co-administration with ketorolac, but not by dexamethasone.In contrast to the acute hypertensive effects of LNP2 infusion, severalepisodes of hypotension (lasting 1-2 hours at a time) were observedfollowing LNP2 infusion, starting about 9 hours after the start ofinfusion (FIG. 3). On each occasion, the average mean arterial pressurewas decreased by 20-30 mmHg, relative to vehicle control, and wasprevented by co-administration with either ketorolac or dexamethasone.

In conclusion, intravenous infusion of 0.3 mg/kg LNP2 over 60 minutes toconscious female minipigs resulted in an acute infusion reaction thatoccurred during and shortly after infusion, and was characterized by anelevation of venous thromboxane B2 concentrations, and an increase inblood pressure. A more delayed infusion reaction occurred approximately8 hours after the end of the infusion, involving several episodes ofhypotension. Co-administration of LNP2 with ketorolac completelyprevented the acute infusion reaction and effectively mitigated the moredelayed hypotension. Co-administration of LNP2 with dexamethasone hadminimal to no effect on the acute infusion reaction, but effectivelyprevented the more delayed hypotension.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A method of ameliorating an infusion reactionassociated with intravenous administration of at least one lipidformulated therapeutic agent in a mammal in need thereof, comprisingadministering to the mammal via injection a therapeutically effectiveamount of a nonsteroidal anti-inflammatory (NSAID) prior to the at leastone lipid formulated therapeutic agent being intravenously administered,wherein the NSAID is ketorolac, wherein the infusion reaction compriseshypertension followed by hypotension and an increase in plasmathromboxane B2 levels, wherein the at least one lipid formulatedtherapeutic agent is formulated in a lipid nanoparticle (LNP), andwherein the lipid formulated therapeutic agent is a nucleic acid.
 2. Themethod of claim 1, wherein the administration of the NSAID begins withinabout 2 hours prior to the administration of the at least one lipidformulated therapeutic agent.
 3. The method of claim 1, wherein theketorolac is administered in a dose of about 15 mg to about 60 mg. 4.The method of claim 3, wherein the ketorolac is administered in a doseof about 30 mg to about 60 mg.
 5. The method of claim 1, wherein the atleast one lipid formulated therapeutic agent is formulated in a lipidnanoparticle (LNP) comprising: a) the at least one therapeutic agent; b)a cationic lipid; and C) a non-cationic lipid.
 6. The method of claim 1,wherein the lipid formulated therapeutic agent is siRNA or mRNA.
 7. Themethod of claim 6, wherein the lipid formulated therapeutic agent issiRNA.
 8. The method of claim 6, wherein the lipid formulatedtherapeutic agent is mRNA.
 9. The method of claim 5, further comprisingadministering an additional therapeutic agent.
 10. The method of claim9, wherein the additional therapeutic agent is dexamethasone.
 11. Themethod of claim 1, wherein the NSAID is administered parenterally,intravenously, intramuscularly or subcutaneously.